DE60224541T2 - USE OF DC-SIGN AND DC-SIGNR TO INHIBIT VIRUS INFECTION WITH HEPATITIS C - Google Patents

Abstract

This invention provides a method of inhibiting HCV infection of a cell susceptible to HCV infection which comprises contacting the cell with an amount of a compound effective to inhibit binding of an HCV envelope glycoprotein to a DC-SIGN protein present on the surface of the cell, so as to thereby inhibit HCV infection of the cell susceptible to HCV infection. This infection provides a method of inhibiting HCV infection of a cell susceptible to HCV infection which comprises contacting the cell with an amount of a compound effective to inhibit binding of an HCV envelope glycoprotein to a DC-SIGNR protein present on the surface of the cell, so as to thereby inhibit HCV infection of the cell susceptible to HCV infection. Compounds of the present invention inhibit HCV infection of cells susceptible to HCV infection. The compounds of the present invention preferably have specificity for preventing or inhibiting infection by HCV and do not inhibit infection by other viruses, such as HIV, that may utilize DC-SIGN or DC-SIGNR for infection. Moreover the compounds of the present invention preferably do not interfere or inhibit members of the immunoglobulin superfamily, in particular, the compounds do not interfere with ICAM-2 or ICAM-3 or with ICAM-2-ilke, or ICAM-3-like molecules.

Description

in the The scope of the present application is indicated by Arabic numerals different publications Referenced. full References to these publications are at the end of the specification immediately before the claims too Find.

Background of the invention

Of the Hepatitis C virus was first identified in 1989 and is responsible for the majority of cases of non-A and non-B hepatitis. [1] The infections are typical chronic and persist for a lifetime; many infected individuals stay over Decades healthy and undisturbed, whereas others develop chronic hepatitis or cirrhosis, the latter in many cases to hepatocellular carcinomas leads. [16] While the screening of blood stores new transfers of the virus drastically reduced, there is still a large amount of infected individuals, which require treatment in the coming decades. Some Reports are from the estimate out that nearly 3% of the world population (including about 4 million people in the United States) are infected with HCV are. [2] There are estimates according to which 170 million people worldwide are infected with HCV are inclusive about 4 million people in the United States. infected Individuals will have a liver disease associated with clinical Effects starting with being asymptomatic carriers, to develop or have active hepatitis and cirrhosis this already developed. Also becomes a chronic infection strongly associated with the development of hepatocellular carcinomas. HCV infection and its clinical sequelae are the principal reasons for liver transplants in the USA. Currently no vaccine available. Some preparations with Interferon-alpha and interferon-alpha-2b plus ribavirin are currently being used for the Treatment of chronic hepatitis C used. [32] The best Long-term reaction rates are combined with a combination of interferon-alpha-2b and ribavirin. However, it succeeds only with a minority of subjects treated with this combination achieve the desired result, d. H. that 6 months after the completion of the treatment no detectable Serum HCV RNA present is. [32] It has not yet been possible for all infected individuals, including the with simultaneous HIV-1 infection, optimal treatment with These agents are established because of the viral dynamics in the Response to treatment data present are sparse. In interferon-alpha and ribavirin are non-specific viral agents, whose action mechanisms have so far been understood only incompletely. she are also associated with severe and life-threatening toxicities, inclusively Neutropenia, hemolytic anemia and severe depression.

It there is an urgent need for new therapeutic agents for fighting the HCV infection. To be a particularly attractive target for the antiviral Therapy is the entry of HCV into target cells, since such inhibitors neither cross the plasma membrane nor modified intracellularly Need to become. Furthermore, viral cell entry is generally rate limiting Step through which conserved structures on the virus and the cell membrane is mediated. Consequently, inhibitors of the viral Cell entry is an effective and permanent suppression of the viral Provide replication.

at the HCV genome is a 9.4 kilobase long, positive-sense, single-stranded RNA molecule which encodes a single polyprotein of ~ 3,000 amino acids. [42] There were characterized a number of isolates and it was found that this one considerable sequence diversity showed. Virus sequences fall into major genotypes (with <70% sequence identity) and further in subgenotypes (with 80-90% Identity) organize. [53] Genotype 1 (subgenotypes 1a and b) prevails in North America, Europe and Japan. [46] There is no clear one Distinctions in terms of pathology associated with the various Genotypes are associated.

Despite the sequence diversity among the isolates, they have many properties in common. The genomic RNA contains a long untranslated 5 'region (NTR) of about 340 nucleotides, followed by a single long open reading frame (ORF) encoding a polyprotein of about 3,000 amino acids. [42] A short 3'-NTR is followed by a polyA sequence and 98 highly conserved nucleotides (the "X" region) .The translation of the RNA is mediated by an IRES element in the 5'-NTR, the polyprotein precursor is further processed to produce at least ten proteins: in the direction from the amino to the carboxy terminus these are designated: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. [19] The C protein is constituted the nucleocapsid, E1 and E2 are transmembrane envelope glycoproteins, the function of p7 is unknown, the different nucleic acid proteins are non-structural proteins with replication functions, and polyprotein cleavage in the structural region (C-p7) is reported in endoplasmic reticulum (ER) catalyzed by cellular signal peptidases ins in the non-structural region (NS2-NS5B) is mediated by HCV-encoded proteinases. NS2 and NS2 constitute a protease which cleaves the NS2 NS3 junction. NS3 is a dual functional protein containing at its amino terminus a serine protease domain responsible for cleavage at the remaining sites in the precursor and an RNA helicase / NTPase domain at its carboxy terminus. It is believed that NS4A enhances or controls the protease activity of NS3, while the functions of NS4B and NS5A are not clear. NS5B is an RNA-dependent RNA polymerase (RdRp) and the catalytic subunit of the replicase for the virus. This enzyme recognizes the 3 'end of the RNA and performs RNA synthesis, producing a minus strand RNA. The 3 'end of the minus strand is then similarly recognized by the RdRp, thereby initiating the synthesis of plus strand RNAs. In the generation of this progeny of viral RNAs, this is bundled into assembly virions. HCV particles bud into the ER and are transported out of the cell by microsomal vesicles. [42]

It are few animal models for an HCV infection. These include the chimpanzees [22] [27] [45], which is an endangered species. One Another model is the SCID-BNX model, by which immunodeficient mice human liver tissue is implanted, which as described with HCV is infected. [54] Studies on viral replication in vitro were largely based so far on the infection of cell lines or of primary hepatic cultures Sera from HCV-infected patients. [4] [5] [23] [24] [26] [29] [44] [51] The levels of viral RNA in these infected cultures but are very low and can can only be detected by PCR. [4] [5] [23] [24] [26] [29] [44] [51] In a significant recent scientific advances have been replaced by Lohmann et al., [30] the structural genes in the genome of a complete subgenotype 1b the neomycin phosphotransferase gene, followed by the IRES of the encephalomyocarditis virus. In the resulting construct was the phosphotransferase gene downstream the HCV 5'-NTR (which containing HCV-IRES), while the HCV non-structural genes are downstream of the IRES of the encephalomyocarditis virus were. RNA was transcribed from this construct and transformed into transfected the human hepatoma cell line Huh-7. After the selection in neomycin, cell lines were obtained which provided a robust replication of the transfected mini genome; viral RNA could through Northern analysis were detected and viral proteins could by means of immunoprecipitation be detected. There is an urgent need for additional Animal models of HCV infection.

Of the Cell entry of HCV into host cells requires attachment of the viral particle at the cell surface, followed by a fusion the viral envelope with the cell membrane. This process is by the viral envelope glycoproteins E1 and E2 mediated. It has been suggested that there are two Proteins designated E1 and E2 (corresponding to amino acids 192-383 and 384-750 the HCV polyprotein), are external proteins of the viral envelope, which bind to the virus are responsible to target cells. HCV-E1 and -E2 were in a number of forms and using a variety of Expression systems recombinantly expressed. Two recent reports described the fusion and the cell entry caused by E1 and E2 ectodomains which were linked to the TM domain of the VSV-G envelope glycoprotein are merged. [28] [49]

In on mammalian cells based expression systems, the molecular weight of mature, in full length present E1 ~ 35 kD and that of E2 is 72 kD. [19] [31] [48] The amino-terminal residues of mature E1 and E2 became experimental certainly. [21] Endoproteolytic processing of the HCV polyprotein converts E1 and E2 are membrane-anchored type 1 proteins. [19] [48] Furthermore, E1 and E2 form non-covalently associated heterodimers, which are referred to below as E1 / E2. [8] [19] [37] [41] Completely processed E1 / E2 heterodimers are not exported to the cell surface, but are retained in the ER, where the HCV budding occurs. [9] [10] [11] [12] [43] Analyzes of N-linked Glycosylation patterns of E1 and E2 also showed that these proteins are held in the ER without circulating through the Golgi apparatus. [12] [34] The ER retention signals are in the TM domains of E1 and E2 located. [6] [7] [14] A substitution of TM domains by E1 and E2 through the TM domains of plasma membrane-associated proteins or the mutation of charged Residues in the TM domains of E1 and E2 resulted in the surface expression of the envelope glycoproteins. [6] [7] [8] [14] Put such modifications of TM domains however, also the E1 / E2 heterodimerization inoperative. [7] [36] The dimerization and ER retention signals of E1 and E2 can not therefore be different from each other be separated. A deletion of the entire TM domain of E1 and E2 results in the secretion of soluble, monomeric ectodomains of Envelope glycoproteins. [12] [13] [35]

To date, two human cellular proteins, receptors for CD81 and low density lipoprotein (LDL), have been implicated as putative receptors mediating HCV cell entry [25] and glycosaminoglycans have been suggested to play a role in non-specific attachment of HCV a cell play. [52] Uses of CD81 in the treatment and diagnosis of HCV infection are described by Abrignani et al. in the international patent application WO 99/18198 disclosed. Studies showed that the recombinant soluble E2 ectodomain binds specifically and with high affinity to human and chimpanzee CD81, but not to CD81 from other species. [15] [20] [38] [39] However, recent studies have questioned these results, including a study showing that CD81 of tamarin monkey, a species refractory to HCV infection, also has high affinity soluble E2 binds. [33] Although a number of studies have defined the structural determinants of human CD81 / E2 interaction, there is still a lack of direct functional evidence for CD81-mediated fusion and cell entry of HCV. In addition, CD81 is expressed on numerous tissues outside the liver, and thus distribution of CD81 in tissue can not explain the cell tropism of HCV. Similarly, no previous studies have shown a direct interaction between LDL receptors and the HCV envelope glycoproteins. [52] Furthermore, the expression of LDL receptors is widespread on tissues other than the liver, and therefore its expression does not explain the tropism of HCV.

at DC-SIGN (Dendrite Cell Special Intercellular Adhesion Molecule-3 Grabbing Nonintegrin, GenBank accession number AF209479) and DC-SIGNR (DC-SIGN-related, GenBank accession number AF245219) are membrane proteins Type II with high sequence homologies (77% identity in amino acids). DC-SIGN will be in big Amounts expressed on dendritic cells; DC-SIGNR will be in large quantities in liver and lymph nodes, but not dendritic cells expressed; and both molecules are expressed on the endometrium and placenta. [40] [47] [3] [17]

The proteins are C-type (calcium dependent) lectins, all of which are known to be required for binding mannose. DC-SIGN and DC-SIGNR bind the HIV-1 surface envelope glycoprotein gp120, which has mannose-rich sugars, and this binding is inhibited by mannan. [47] [3] [17] Both DC-SIGN and DC-SIGNR bind infectious HIV-1 particles and promote transfection of susceptible T cells into trans. [40] [47] [3] The European patent applications EP 1046651 A1 and EP 1086137 A1 describe the use of DC-SIGN in compositions and methods for inhibiting HIV-1 infection. The content of these applications is hereby incorporated by reference in their entirety.

As DC-SIGN and DC-SIGNR also bind the lectin Galanthus nivalis (GNA-lectin) from snowdrop bulbs extremely readily Carbohydrates and glycoproteins with high mannose structures. Especially GNA lectin readily binds HIV-1 envelope glycoproteins. [18] [50] Besides catches GNA the HCV envelope glycoproteins a [13], which contain high mannose carbohydrates. On the Based on these results, we realized that DC-SIGN and DC-SIGNR are extremely willing HCV envelope glycoproteins bind and therefore serve as receptors for the virus.

So far as we know, there has been no correlation between DC-SIGN, DC-SIGNR and HCV infection produced. DC-SIGN and DC-SIGNR are also able to to mediate the internalization as it does for the cell entry and a Infection by HCV, but not by HIV-1, is required. Furthermore, DC-SIGNR is particularly expressed in large quantities in the liver, the primary Target organ for an HCV infection. Because the ability from DC-SIGN and especially DC-SIGNR, as receptors for HCV too act, as yet unrecognized, offers this discovery the possibility, treat HCV infection with therapies or vaccines or to prevent what the specific interaction between Block HCV and these receptors.

Summary of the invention

The The present invention discloses a method for inhibiting a HCV infection one for susceptible to HCV infection A cell comprising contacting the cell with an amount a compound which is sufficiently effective to bind an HCV envelope glycoprotein to a on the surface of the Cell to inhibit present DC-SIGN protein, thereby causing an HCV infection for susceptible to HCV infection Cell is inhibited. In one embodiment, the compound inhibits no HCV binding to a DC-SIGNR protein. In a further embodiment the compound does not inhibit HIV infection of a person susceptible to HIV infection Cell. In a further embodiment The compound does not block ICAM adhesion.

The present invention provides a method for inhibiting HCV infection of a cell susceptible to HCV infection, comprising contacting the cell with an amount of a compound that is sufficiently effective to bind an HCV envelope glycoprotein to an on the surface to inhibit the DC-SIGNR protein present in the cell to thereby inhibit HCV infection of the cell susceptible to HCV infection. In one embodiment, the compound does not inhibit HCV binding to a DC-SIGN protein. In another embodiment, the compound does not inhibit HIV infection of a cell susceptible to HIV infection. In another embodiment, the compound does not block ICAM adhesion.

The The present invention discloses a method for inhibiting a HCV infection of a target cell whose susceptibility to HCV infection elevated when HCV binds to a DC-SIGN protein expressing cell, comprising contacting the DC-SIGN protein expressing Cell with an amount of a compound which is sufficiently effective is the binding of an HCV envelope glycoprotein to inhibit a DC-SIGN protein, thereby inhibiting HCV infection of the target cell to inhibit. In one embodiment the compound does not inhibit HCV binding to a DC-SIGNR protein. In a further embodiment the compound does not inhibit HIV infection of a person susceptible to HIV infection Cell. In a further embodiment The compound does not block ICAM adhesion.

The The present invention provides a method for inhibition a HCV infection a target cell whose receptivity for one HCV infection increased when HCV binds to a DC-SIGNR protein expressing cell, comprising contacting the DC-SIGNR protein expressing Line with an amount of a compound which is sufficiently effective is the binding of an HCV envelope glycoprotein to inhibit a DC-SIGNR protein, thereby inhibiting HCV infection of the target cell to inhibit. In one embodiment the compound does not inhibit HCV binding to a DC-SIGN protein. In a further embodiment the compound does not inhibit HIV infection of a person susceptible to HIV infection Cell. In a further embodiment The compound does not block ICAM adhesion.

A embodiment The invention involves the use of one of the previously described Method whereby the subject may be pregnant. The connection can before, while or after delivery to such a subject. In a further embodiment invention, the compound blocks the placental transmission from HCV to a fetus.

The The present invention provides a method which comprises a HCV infection of for susceptible to HCV infection Cells of a subject by means of a method described herein inhibited.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGN-Protein, um alle Bindungsstellen für das DC-SIGN-Protein auf dem immobilisierten HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche die Bindung des DC-SIGN-Proteins an das immobilisierte HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGN-Protein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges DC-SIGN-Protein aus dem Komplex verdrängt wird, wobei eine Verdrängung von DC-SIGN-Protein aus dem Komplex anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGN protein to saturate all binding sites for the DC-SIGN protein on the immobilized HCV envelope glycoprotein under conditions involving binding of the DC-SIGN protein allow the immobilized HCV envelope glycoprotein to form a complex;
• c) removing unbound DC-SIGN protein;
• d) contacting the complex with the compound; and
• e) determining whether any DC-SIGN protein is displaced from the complex, wherein displacement of DC-SIGN protein from the complex indicates that the compound binds to the HCV envelope glycoprotein, thereby determining that it is in the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGNR-Protein, um alle Bindungsstellen für das DC-SIGNR-Protein auf dem immobilisierten HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche die Bindung des DC-SIGNR-Proteins an das immobilisierte HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges DC-SIGNR-Protein aus dem Komplex verdrängt wird, wobei eine Verdrängung von DC-SIGNR-Protein aus dem Komplex anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGNR protein to saturate all binding sites for the DC-SIGNR protein on the immobilized HCV envelope glycoprotein under conditions involving binding of the DC-SIGNR protein allow the immobilized HCV envelope glycoprotein to form a complex;
• c) removing unbound DC-SIGNR protein;
• d) contacting the complex with the compound; and
• e) determining whether any DC-SIGNR protein is displaced from the complex, wherein displacement of DC-SIGNR protein from the complex indicates that the compound binds to the HCV envelope glycoprotein, thereby determining that it is in the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGN-Proteins mit einer ausreichenden Menge an detektierbarem HCV-Hüllglykoprotein, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGN-Protein unter Bedingungen zu saturieren, welche die Bindung des immobilisierten DC-SIGN-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung von HCV-Hüllglykoprotein aus dem Komplex anzeigt, dass die Verbindung an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the immobilized DC-SIGN protein with a sufficient amount of detectable HCV envelope glycoprotein to saturate all binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGN protein under conditions which inhibit binding of the immobilized DC-SIGN protein. Allow protein to the HCV envelope glycoprotein, so that a complex is formed;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with the compound; and
• e) determining whether any HCV envelope glycoprotein is displaced from the complex, wherein displacement of HCV envelope glycoprotein from the complex indicates that the compound binds to the DC-SIGN protein to thereby determine that the HCV envelope glycoprotein is out of the complex Compound is a compound which is capable of inhibiting a HCV infection of the cell.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGNR-Proteins mit einer ausreichenden Menge an detektierbarem HCV-Hüllglykoprotein, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGNR-Protein unter Bedingungen zu saturieren, welche die Bindung des immobilisierten DC-SIGNR-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung von HCV-Hüllglykoprotein aus dem Komplex anzeigt, dass die Verbindung an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the immobilized DC-SIGNR protein with a sufficient amount of detectable HCV envelope glycoprotein to saturate all binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGNR protein under conditions which inhibit binding of the immobilized DC-SIGNR protein. Allow protein to the HCV envelope glycoprotein, so that a complex is formed;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with the compound; and
• e) determining whether any HCV envelope glycoprotein is displaced from the complex, wherein displacement of HCV envelope glycoprotein from the complex indicates that the compound binds to the DC-SIGNR protein to thereby determine that the HCV envelope glycoprotein is out of the complex Compound is a compound which is capable of inhibiting a HCV infection of the cell.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGN-Protein, um alle Bindungsstellen für das DC-SIGN-Protein auf dem HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche eine Bindung des DC-SIGN-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGN-Protein;
• c) Messen der Menge an DC-SIGN-Protein, welches an das HCV-Hüllglykoprotein in dem Komplex gebunden wird;
• d) Inkontaktbringen des Komplexes mit der Verbindung, so dass DC-SIGN-Protein aus dem Komplex verdrängt wird;
• e) Messen der Menge an DC-SIGN-Protein, welches in der Anwesenheit der Verbindung an die Verbindung gebunden wird; und
• f) Vergleichen der Menge von DC-SIGN-Protein, welches in Schritt e) an das HCV-Hüllglykoprotein gebunden wurde, mit der in Schritt c) gemessenen Menge, wobei eine reduzierte in Schritt e) gemessene Menge anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGN protein to saturate all binding sites for the DC-SIGN protein on the HCV envelope glycoprotein under conditions involving binding of the DC-SIGN protein to the DCSIGN protein Allow HCV envelope glycoprotein to form a complex;
• b) removing unbound DC-SIGN protein;
• c) measuring the amount of DC-SIGN protein bound to the HCV envelope glycoprotein in the complex;
• d) contacting the complex with the compound such that DC-SIGN protein is displaced from the complex;
• e) measuring the amount of DC-SIGN protein bound to the compound in the presence of the compound; and
• f) comparing the amount of DC-SIGN protein bound to the HCV envelope glycoprotein in step e) with the amount measured in step c), wherein a reduced amount measured in step e) indicates that the compound is attached to the HCV envelope glycoprotein HCV envelope glycoprotein binds to thereby determine that the compound is a compound capable of inhibiting HCV infection of the cell.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGNR-Protein, um alle Bindungsstellen für das DC-SIGNR-Protein auf dem HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche eine Bindung des DC-SIGNR-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• c) Messen der Menge an DC-SIGNR-Protein, welches an das HCV-Hüllglykoprotein in dem Komplex gebunden wird;
• d) Inkontaktbringen des Komplexes mit der Verbindung, so dass DC-SIGNR-Protein aus dem Komplex verdrängt wird;
• e) Messen der Menge an DC-SIGNR-Protein, welches in der Anwesenheit der Verbindung an die Verbindung gebunden wird; und
• f) Vergleichen der Menge von DC-SIGNR-Protein, welches in Schritt e) an das HCV-Hüllglykoprotein gebunden wurde, mit der in Schritt c) gemessenen Menge, wobei eine reduzierte in Schritt e) gemessene Menge anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch die Verbindung als eine Verbindung zu identifizieren, welche dazu in der Lage ist, eine HCV-Infektion einer Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGNR protein to saturate all binding sites for the DC-SIGNR protein on the HCV envelope glycoprotein under conditions involving binding of the DC-SIGNR protein to the DCSIGNR protein Allow HCV envelope glycoprotein to form a complex;
• b) removing unbound DC-SIGNR protein;
• c) measuring the amount of DC-SIGNR protein bound to the HCV envelope glycoprotein in the complex;
• d) contacting the complex with the compound such that DC-SIGNR protein is displaced from the complex;
• e) measuring the amount of DC-SIGNR protein bound to the compound in the presence of the compound; and
• f) comparing the amount of DC-SIGNR protein bound to the HCV envelope glycoprotein in step e) with the amount measured in step c), wherein a reduced amount measured in step e) indicates that the compound is attached to the HCV envelope glycoprotein HCV envelope glycoprotein binds to thereby identify the compound as a compound capable of inhibiting HCV infection of a cell.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGN-Protein unter Bedingungen, welche die Bindung des DC-SIGN-Proteins an das immobilisierte HCV-Hüllglykoprotein in der Abwesenheit der Verbindung zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGN-Protein;
• d) Vergleichen der Menge an detektierbarem DC-SIGN-Protein, welche in der Anwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGN-Protein, welche in der Abwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an DC-SIGN-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with the compound and detectable DC-SIGN protein under conditions which permit binding of the DC-SIGN protein to the immobilized HCV envelope glycoprotein in the absence of the compound to form a complex;
• c) removing unbound DC-SIGN protein;
• d) comparing the amount of detectable DC-SIGN protein bound in the presence of the compound to the immobilized HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGN protein immobilized in the absence of the compound HCV envelope glycoprotein binds;
• e) wherein a reduced amount of DC-SIGN protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGN protein to thereby determine that the compound is a A compound which is capable of inhibiting a HCV infection of the cell.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGNR-Protein unter Bedingungen, welche die Bindung des DC-SIGNR-Proteins an das immobilisierte HCV-Hüllglykoprotein in der Abwesenheit der Verbindung zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• d) Vergleichen der Menge an detektierbarem DC-SIGNR-Protein, welche in der Anwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGNR-Protein, welche in der Abwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an DC-SIGNR-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with the compound and detectable DC-SIGNR protein under conditions permitting binding of the DC-SIGNR protein to the immobilized HCV envelope glycoprotein in the absence of the compound to form a complex;
• c) removing unbound DC-SIGNR protein;
• d) comparing the amount of detectable DC-SIGNR protein bound in the presence of the compound to the immobilized HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGNR protein immobilized in the absence of the compound HCV envelope glycoprotein binds;
• e) wherein a reduced amount of DC-SIGNR protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that the compound is a A compound which is capable of inhibiting a HCV infection of the cell.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGN-Proteins mit der Verbindung und detektierbarem HCV-Hüllglykoprotein unter Bedingungen, welche die Bindung des immobilisierten DC-SIGN-Proteins an das HCV-Hüllglykoprotein in der Abwesenheit der Verbindung zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Vergleichen der Menge an detektierbarem HCV-Hüllglykoprotein, welche in der Anwesenheit der Verbindung an das immobilisierte DC-SIGN-Protein in dem Komplex gebunden wird mit der Menge an detektierbarem HCV-Hüllglykoprotein, welche in der Abwesenheit der Verbindung an das immobilisierte DC-SIGN-Protein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an HCV-Hüllglykoprotein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the immobilized DC-SIGN protein with the compound and detectable HCV envelope glycoprotein under conditions permitting binding of the immobilized DC-SIGN protein to the HCV envelope glycoprotein in the absence of the compound to form a complex;
• c) removing unbound HCV envelope glycoprotein;
• d) comparing the amount of detectable HCV envelope glycoprotein bound in the complex in the presence of the compound to the immobilized DC-SIGN protein in the complex with the amount of detectable HCV envelope glycoprotein which in the absence of the compound binds to the immobilized DC SIGN protein binds;
• e) wherein a reduced amount of HCV envelope glycoprotein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGN protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGNR-Proteins mit der Verbindung und detektierbarem HCV-Hüllglykoprotein unter Bedingungen, welche die Bindung des immobilisierten DC-SIGNR-Proteins an das HCV-Hüllglykoprotein in der Abwesenheit der Verbindung zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Vergleichen der Menge an detektierbarem HCV-Hüllglykoprotein, welche in der Anwesenheit der Verbindung an das immobilisierte DC-SIGNR-Protein in dem Komplex gebunden wird mit der Menge an detektierbarem HCV- Hüllglykoprotein, welche in der Abwesenheit der Verbindung an das immobilisierte DC-SIGNR-Protein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an HCV-Hüllglykoprotein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the immobilized DC-SIGNR protein with the compound and detectable HCV envelope glycoprotein under conditions permitting binding of the immobilized DC-SIGNR protein to the HCV envelope glycoprotein in the absence of the compound to form a complex;
• c) removing unbound HCV envelope glycoprotein;
• d) comparing the amount of detectable HCV envelope glycoprotein bound in the presence of the compound to the immobilized DC-SIGNR protein in the complex with the amount of detectable HCV envelope glycoprotein which, in the absence of the compound, is immobilized to the DC SIGNR protein binds;
• e) wherein a reduced amount of HCV envelope glycoprotein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGN-Protein unter Bedingungen, welche die Bindung des DC-SIGN-Proteins an das HCV-Hüllglykoprotein in der Abwesenheit der Verbindung zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGN-Protein;
• c) Vergleichen der Menge an detektierbarem DC-SIGN-Protein, welches in der Anwesenheit der Verbindung an das HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGN-Protein, welches in der Abwesenheit der Verbindung an die Verbindung bindet; wobei eine reduzierte in der Anwesenheit der Verbindung gemessene Menge an DC-SIGN-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with the compound and detectable DC-SIGN protein under conditions which allow the binding of the DC-SIGN protein to the HCV envelope glycoprotein in the absence of the compound to form a complex;
• b) removing unbound DC-SIGN protein;
• c) comparing the amount of detectable DC-SIGN protein bound in the presence of the compound to the HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGN protein which binds to the compound in the absence of the compound ; wherein a reduced amount of DC-SIGN protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGN protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGNR-Protein unter Bedingungen, welche die Bindung des DC-SIGNR-Proteins an das HCV-Hüllglykoprotein in der Abwesenheit der Verbindung zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• c) Vergleichen der Menge an detektierbarem DC-SIGNR-Protein, welches in der Anwesenheit der Verbindung an das HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGNR-Protein, welches in der Abwesenheit der Verbindung an die Verbindung bindet; wobei eine reduzierte in der Anwesenheit der Verbindung gemessene Menge an DC-SIGNR-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with the compound and detectable DC-SIGNR protein under conditions permitting binding of the DC-SIGNR protein to the HCV envelope glycoprotein in the absence of the compound to form a complex;
• b) removing unbound DC-SIGNR protein;
• c) comparing the amount of detectable DC-SIGNR protein bound in the presence of the compound to the HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGNR protein which binds to the compound in the absence of the compound ; wherein a reduced amount of DC-SIGNR protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Identifikation einer Verbindung, welche eine HCV-Infektion einer Zelle gemäß einem hierin beschriebenen Verfahren inhibiert;
• b) Gewinnung der Verbindung; und
• c) Vermischen der auf diese Weise identifizierten Verbindung oder eines Homologs oder eines Derivats davon mit einem Träger, um dadurch eine Zusammensetzung zu erhalten.

The present invention provides a method for obtaining a composition comprising:

• a) identification of a compound which inhibits HCV infection of a cell according to a method described herein;
• b) obtaining the compound; and
• c) mixing the thus identified compound or a homologue or a derivative thereof with a carrier to thereby obtain a composition.

The The present invention discloses the use of an effective amount a compound which is capable of binding a HCV envelope glycoprotein to one on the surface to inhibit cells of a subject present DC-SIGN protein, for the Preparation of a medicament for treatment or prevention a liver disease in a subject.

The The present invention provides the use of an effective amount a compound that is capable of binding an HCV envelope glycoprotein to one on the surface to inhibit the cells of a subject existing DC-SIGNR protein, for the Preparation of a medicament for treatment or prevention a liver disease in a subject.

The The present invention discloses the use of an effective amount a compound which is capable of binding a HCV envelope glycoprotein to one on the surface to inhibit cells of a subject present DC-SIGN protein, for the Preparation of a medicament for treatment or prevention of a hepatocellular Carcinoma in a subject.

The present invention provides the use of an effective amount of the compound, wel It is capable of inhibiting the binding of an HCV envelope glycoprotein to a DC-SIGNR protein present on the surface of the cells of a subject for the manufacture of a medicament for the treatment or prevention of hepatocellular carcinoma in a subject.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGN-Proteins mit einer Menge an HCV-Hüllglykoprotein, welche ausreichend ist, um alle oder einen Teil der Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGN-Protein zu saturieren, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• e) Entfernen von nicht gebundener Probe; und
• f) Bestimmung, ob Antikörper an das HCV-Hüllglykoprotein gebunden wurde, wobei die Anwesenheit von anti-HCV-Antikörpern dadurch eine HCV-Infektion des Subjekts diagnostiziert.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the immobilized DC-SIGN protein with an amount of HCV envelope glycoprotein sufficient to saturate all or a portion of the binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGN protein to form a complex becomes;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with a suitable sample obtained from the subject;
• e) removing unbound sample; and
• f) determining whether antibody has been bound to the HCV envelope glycoprotein, wherein the presence of anti-HCV antibodies thereby diagnoses HCV infection of the subject.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGNR-Proteins mit einer Menge an HCV-Hüllglykoprotein, welche ausreichend ist, um alle oder einen Teil der Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGNR-Protein zu saturieren, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• e) Entfernen von nicht gebundener Probe; und
• f) Bestimmung, ob Antikörper an das HCV-Hüllglykoprotein gebunden wurde, wobei die Anwesenheit von anti-HCV-Antikörpern dadurch eine HCV-Infektion des Subjekts diagnostiziert.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the immobilized DC-SIGNR protein with an amount of HCV envelope glycoprotein sufficient to saturate all or a portion of the binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGNR protein to form a complex becomes;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with a suitable sample obtained from the subject;
• e) removing unbound sample; and
• f) determining whether antibody has been bound to the HCV envelope glycoprotein, wherein the presence of anti-HCV antibodies thereby diagnoses HCV infection of the subject.

• a) Inkontaktbringen von immobilisiertem DC-SIGN-Protein mit einer Menge an HCV-Hüllglykoprotein, welche ausreichend ist, um alle oder einen Teil der Bindungsstellen für das HCV-Hüllglykoprotein auf dem DC-SIGN-Protein zu saturieren, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• c) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• d) Entfernen von nicht gebundener Probe; und
• e) Bestimmung, ob Antikörper an das HCV-Hüllglykoprotein gebunden wurde, wobei die Anwesenheit von anti-HCV-Antikörpern dadurch eine HCV-Infektion des Subjekts diagnostiziert.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) contacting immobilized DC-SIGN protein with an amount of HCV envelope glycoprotein sufficient to saturate all or part of the binding sites for the HCV envelope glycoprotein on the DC-SIGN protein to form a complex ;
• b) removing unbound HCV envelope glycoprotein;
• c) contacting the complex with a suitable sample obtained from the subject;
• d) removing unbound sample; and
• e) determining whether antibody has been bound to the HCV envelope glycoprotein, wherein the presence of anti-HCV antibodies thereby diagnoses HCV infection of the subject.

• a) Inkontaktbringen von immobilisiertem DC-SIGNR-Protein mit einer Menge an HCV-Hüllglykoprotein, welche ausreichend ist, um alle oder einen Teil der Bindungsstellen für das HCV-Hüllglykoprotein auf dem DC-SIGNR-Protein zu saturieren, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• c) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• d) Entfernen von nicht gebundener Probe; und
• e) Bestimmung, ob Antikörper an das HCV-Hüllglykoprotein gebunden wurde, wobei die Anwesenheit von anti-HCV-Antikörpern dadurch eine HCV-Infektion des Subjekts diagnostiziert.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) contacting immobilized DC-SIGNR protein with an amount of HCV envelope glycoprotein sufficient to saturate all or part of the binding sites for the HCV envelope glycoprotein on the DC-SIGNR protein to form a complex ;
• b) removing unbound HCV envelope glycoprotein;
• c) contacting the complex with a suitable sample obtained from the subject;
• d) removing unbound sample; and
• e) determining whether antibody has been bound to the HCV envelope glycoprotein, wherein the presence of anti-HCV antibodies thereby diagnoses HCV infection of the subject.

The The present invention discloses an antibody or a part thereof which is capable of binding a DC-SIGN protein to an HCV envelope glycoprotein to inhibit, said antibody binds to an epitope, which is within a region of the DC-SIGN protein, wherein said region of the DC-SIGN protein to an HCV envelope glycoprotein binds. In one embodiment the antibody inhibits HCV binding to a DC-SIGNR protein is not. In another embodiment the antibody inhibits an HIV infection one for an HIV infection susceptible Cell not. In another embodiment, the antibody blocks the ICAM adhesion Not.

The present invention discloses an antibody or portion thereof which is capable of inhibiting the binding of a DC-SIGNR protein to an HCV envelope glycoprotein, said An antibody binds to an epitope which lies within a region of the DC-SIGNR protein, wherein said region of the DC-SIGNR protein binds to an HCV envelope glycoprotein. In one embodiment, the antibody does not inhibit HCV binding to a DC-SIGN protein. In another embodiment, the antibody does not inhibit HIV infection of a cell susceptible to HIV infection. In another embodiment, the antibody does not block ICAM adhesion.

The The present invention discloses an antibody or a part thereof which is capable of binding a DC-SIGN protein to an HCV envelope glycoprotein to inhibit, said antibody binds to an epitope, which is within a region of the HCV envelope glycoprotein, wherein the said region of the HCV envelope glycoprotein binds to a DC-SIGN protein. In one embodiment, the antibody inhibits the HCV binding to a DC-SIGNR protein is not. In a further embodiment the antibody inhibits an HIV infection one for an HIV infection susceptible Cell not. In another embodiment, the antibody blocks the ICAM adhesion Not.

The The present invention discloses an antibody or a part thereof which is capable of binding a DC-SIGNR protein to an HCV envelope glycoprotein to inhibit, said antibody binds to an epitope, which is within a region of the HCV envelope glycoprotein, wherein the said region of the HCV envelope glycoprotein binds to a DC-SIGNR protein. In one embodiment, the antibody inhibits the HCV binding to a DC-SIGN protein is not. In a further embodiment the antibody inhibits an HIV infection one for an HIV infection susceptible Cell not. In another embodiment, the antibody blocks the ICAM adhesion Not.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGN protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence comprising the sequence of at least one section an extracellular Domain of one DC-SIGN protein corresponds, said portion to an HCV envelope glycoprotein binds. In one embodiment does not bind the polypeptide to an HIV envelope glycoprotein. In another Embodiment inhibited the polypeptide is an HIV infection of a susceptible to HIV infection Cell not. In another embodiment, the polypeptide blocks the ICAM adhesion Not.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGNR protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence comprising the sequence of at least one section an extracellular domain a DC-SIGNR protein corresponds, said portion to an HCV envelope glycoprotein binds. In one embodiment does not bind the polypeptide to an HIV envelope glycoprotein. In a further embodiment the polypeptide inhibits HIV infection of one for HIV infection receptive Cell not. In another embodiment, the polypeptide blocks the ICAM adhesion Not.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGN protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence comprising the sequence of at least one section an extracellular Domain of one HCV envelope glycoprotein corresponds, wherein said section binds to a DC-SIGN protein.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGNR protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence comprising the sequence of at least one section an extracellular domain an HCV envelope glycoprotein corresponds, said portion to a DC-SIGNR protein binds.

The The present invention discloses a non-peptidyl agent which capable of binding a DC-SIGN protein to a HCV envelope glycoprotein to inhibit said nonpeptidyl agent to an epitope binds which lies within a region of the DC-SIGN protein, wherein said region of the DC-SIGN protein is linked to an HCV envelope glycoprotein binds. In one embodiment The non-peptidyl agent inhibits HCV binding to a DC-SIGNR protein Not. In a further embodiment The non-peptidyl agent inhibits HIV infection of a person susceptible to HIV infection Cell not. In another embodiment, the nonpeptidyl agent blocks the ICAM adhesion Not.

The present invention discloses a non-peptidyl agent which is capable of inhibiting the binding of a DC-SIGNR protein to an HCV envelope glycoprotein, said nonpeptidyl agent binding to an epitope which is within a region of the DC. SIGNR protein, wherein said region of the DC-SIGNR protein binds to an HCV envelope glycoprotein. In one embodiment, the non-pep inhibits tidyl agent does not interfere with HCV binding to a DC-SIGN protein. In another embodiment, the non-peptidyl agent does not inhibit HIV infection of a cell susceptible to HIV infection. In another embodiment, the nonpeptidyl agent does not block ICAM adhesion.

The The present invention discloses a non-peptidyl agent which capable of binding a DC-SIGN protein to a HCV envelope glycoprotein to inhibit, wherein the non-peptidyl agent at least one Section of an extracellular domain an HCV envelope glycoprotein binds, said portion binding to a DC-SIGN protein. In one embodiment The non-peptidyl agent inhibits HCV binding to a DC-SIGNR protein Not. In a further embodiment The nonpeptidyl agent inhibits HIV infection of one for HIV infection receptive Cell not. In another embodiment, the nonpeptidyl agent blocks the ICAM adhesion Not.

The The present invention discloses a non-peptidyl agent which is capable of binding a DC-SIGNR protein to a HCV envelope glycoprotein to inhibit, wherein the non-peptidyl agent at least one Section of an extracellular domain an HCV envelope glycoprotein binds, said portion binds to a DC-SIGNR protein. In one embodiment The non-peptidyl agent inhibits HCV binding to a DC-SIGN protein Not. In a further embodiment The nonpeptidyl agent inhibits HIV infection of one for HIV infection receptive Cell not. In another embodiment, the nonpeptidyl agent blocks the ICAM adhesion Not.

The The present invention discloses a composition comprising a antibody or a part thereof, a polypeptide described herein and / or Non-peptide agent and a carrier. In one embodiment The composition further includes mannan, a calcium chelator or combinations thereof.

Brief description of the figures:

1 :

amino acid sequence for DC-SIGN from Homo sapiens as set forth in GenBank No. AAK20997 (SEQ ID NUMBER 1).

2 :

amino acid sequence for DC-SIGNR from Homo sapiens as set forth in GenBank No. AAG13848 (SEQ ID NO. 2).

3 :

amino acid sequence for the Hepatitis C virus polyprotein gene as set forth in GenBank No. AF009606 (SEQ ID NO: 3).

4 :

characterization of HeLa DC SIGN and HeLa DC SIGNR cell lines using of antibodies, which for DC-SIGN (507 (D)), DC-SIGNR (604 (L)) or for both molecules (612 (X)) are specific.

5 :

DC-SIGN and DC-SIGNR transfectants bind HCV E2. (A) HeLa-DC-SIGN, (B) HeLa-DC-SIGNR and (C) parental HeLa cells were allowed to to bind with HCV-E2 coated beads, which by conjugation with a panel of anti-E2 mABs were manufactured. The adhesion was quantitated by FACS analysis and blocked by mannan (20 μg / ml); a representative Experiment of three is shown.

6 :

The effect of mABs on the adhesion of HCV-E2 to DC-SIGN or DC-SIGNR. HeLa cells expressing DC-SIGN or DC-SIGNR were incubated with mABs or with mannan as described and H53-conjugated E2 beads were added in a ratio of 20: 1 beads / cell. The bond was quantified by fluorescence using a FACScan instrument and the results were normalized to control level (IgG2a).

Detailed description the invention

The The present invention discloses a method for inhibiting a HCV infection one for susceptible to HCV infection A cell comprising contacting the cell with an amount on a compound which is sufficiently effective to bind an HCV envelope glycoprotein to one on the surface to inhibit the DC-SIGN protein present in the cell, thereby HCV infection for susceptible to HCV infection Cell to inhibit. The present invention provides a method for inhibiting HCV infection of a person susceptible to HCV infection A cell ready comprising contacting the cell with a Amount of a compound which is sufficiently effective to the Binding of an HCV envelope glycoprotein to one on the surface the cell present DC-SIGNR protein to thereby inhibit HCV infection for HCV infection receptive Cell to inhibit.

For a HCV infection receptive Cells can Virus by DC-SIGN and / or DC SIGNR molecules tie. Furthermore can Cells that are not receptive for one HCV infection is to bind virus through DC-SIGN and / or DC-SIGNR molecules. Bound virus is then transferred to a second susceptible target cell in trans. Accordingly, the present invention provides a method for inhibiting the initial attachment of virus to a DC-SIGN and / or DC-SIGNR expressing non-susceptible Cell ready; this leads to then to prevention a subsequent infection of the susceptible target cell. The present Invention discloses a method for inhibiting HCV infection a target cell whose receptivity for one HCV infection increased when HCV binds to a second cell at which it is is a DC-SIGN protein expressing cell, the said Method of contacting the DC-SIGN protein expressing Cell comprising an amount of a compound which is sufficiently effective is the binding of an HCV envelope glycoprotein to inhibit a DC-SIGN protein, thereby inhibiting HCV infection of the target cell to inhibit. The present invention provides a method for Inhibition of HCV infection of a target cell ready, their susceptibility for one HCV infection increased when HCV binds to a second cell at which it is is a DC SIGNR protein expressing cell, wherein the said method involves contacting the DC-SIGN protein expressing Cell comprising an amount of a compound which is sufficient is effective for binding an HCV envelope glycoprotein to a DC-SIGN protein to thereby inhibit HCV infection of the target cell.

The The present invention discloses a method for inhibiting a HCV infection of a target cell which has no on its surface DC SIGN and / or DC SIGNR receptor comprising contacting with a second cell, which on their surface a DC-SIGN and / or DC-SIGNR receptor expressed, with a Amount of a compound described herein which is sufficient is effective in binding HCV to the DC-SIGN and / or DC-SIGNR receptors to thereby inhibit HCV infection of the first target cell to inhibit in trans. In one embodiment of the present method the target cell is present in a subject and bringing into contact is done by administering the compound to the subject. In a embodiment is the target cell, which does not have the DC-SIGN and / or the DC-SIGNR receptor expressed, near the second cell, the DC-SIGN- and / or the DC-SIGNR receptor. In one embodiment border the target cell and the second cell together. In a another embodiment the target cell is not near the second cell. In a embodiment the target cell and the second cell are less than 1 Å apart away, at least 1 Å apart away, at least 10 Å from each other away, at least 100 Å apart removed, at least 1 nm apart, at least 10 nm away from each other, at least 100 nm apart, at least 1 μm from each other removed, at least 10 microns away from each other, at least 100 microns apart, at least 1 mm apart, at least 1 cm apart, at least 10 cm apart and at least 100 cm apart away, at least 1 m apart.

As used herein, "HCV" refers to the hepatitis C virus HCV includes, but is not limited to, extracellular virus particles and the forms of HCV that are associated with HCV infected cells or found in HCV infected cells. As used herein, a "cell expressing HCV envelope glycoprotein on its surface" may also be referred to as an "HCV envelope glycoprotein cell." As used herein, "HCV infection" refers to the introduction of HCV genetic information into a target cell, as by fusion of the target cell membrane with HCV or with an HCV envelope glycoprotein cell. The target cell may be a body cell of a subject. In one embodiment, the target cell is a body cell of a subject, such as a human subject. As used herein, "inhibition of HCV infection" refers to the reduction in the amount of genetic information of HCV, Which is introduced into a population of target cells, compared to the amount which z. B. without an inhibiting agent would be introduced. As used herein, "inhibiting" means that the amount is reduced compared to the amount that would be present in a control sample, for example, a control sample may be a sample that does not contain the inhibitory agent and in which, accordingly, no inhibition of HCV infection would occur In a preferred embodiment, "inhibiting" means that the amount is reduced by 100%. As used herein, "fusion" refers to the joining or unification of the lipid bilayer membranes found on mammalian cells or viruses such as HCV, which differs from the attachment of HCV to a target cell HCV glycoprotein is mediated to a ligand present on the surface of a cell susceptible to HCV infection As used herein, such a ligand includes DC-SIGN or DC-SIGNR As used herein, "cell membrane fusion refers to a HCV infection susceptible cell with the HCV envelope glycoprotein ⁺ cell membrane "the hydrophobic connection and integration of the cell membrane of the infection-susceptible cell with the HCV envelope glycoprotein ⁺ cell to form a hybrid membrane comprising components of both cell membranes. As used herein, "attachment" refers to the process mediated by the binding of the HCV envelope glycoprotein to a ligand present on the surface of a cell susceptible to HCV infection. As used herein, "inhibiting the fusion of an HCV infection" Envelope glycoprotein ⁺ cell having a cell susceptible to HCV infection "(a) a reduction in the fusion rate of a cell membrane of a cell susceptible to HCV infection to a cell membrane of an HCV envelope glycoprotein ⁺ cell by at least 5%, or (b) a reduction in the total extent of fusion of a cell membrane of a cell susceptible to HCV infection to an HCV envelope glycoprotein ⁺ cell membrane at the time of completion of fusion by at least 5%. As used herein, "rate of cell membrane fusion" refers to the total extent of fused cell membrane per unit time. As used herein, "time of completion of fusion" means the time at which the entire fusion of cell membranes from cells susceptible to HCV infection with HCV envelope glycoprotein ⁺ cell membrane, which is possible overall. As used herein, a "cell susceptible to HCV infection" may also be referred to as a "target cell" and includes cells capable of being infected or fused by HCV, or HCV-infected cells , As used herein, the term "cell" includes a biological cell, e.g., a HeLa cell, and a non-biological cell, e.g., a lipid vesicle (e.g., a phospholipid vesicle) or a virion.

In one embodiment of the methods described herein, the compound is an antibody or part of an antibody. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a polyclonal antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a chimeric antibody. In one embodiment, the part of the antibody comprises a light chain of the antibody. In one embodiment, the part of the antibody comprises a heavy chain of the antibody. In one embodiment, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment, the portion of the antibody comprises an F (ab ') ₂ portion of the antibody. In one embodiment, the portion of the antibody comprises an Fd portion of the antibody. In one embodiment, the portion of the antibody comprises an Fv portion of the antibody. In one embodiment, the portion of the antibody comprises a variable domain of the antibody. In one embodiment, the portion of the antibody comprises one or more CDR domains of the antibody.

In an embodiment the method described herein is the compound to a polypeptide. In one embodiment it is the compound is a peptide. In one embodiment it is the compound is an oligopeptide.

In an embodiment the method described herein is the compound to a nonpeptidyl agent. In one embodiment, it is the nonpeptidyl agent is a carbohydrate. Such a carbohydrate may be any carbohydrate known to those skilled in the art, including, however not limited on: mannose, mannan or methyl α-D-mannopyranoside. In one embodiment the method described herein is the compound around a little molecule or a molecule low molecular weight. In one embodiment, the compound a molecular weight of less than 500 daltons.

In an embodiment The method described herein is the HCV envelope glycoprotein an HCV E1 envelope glycoprotein. In one embodiment In the method described herein, the HCV envelope glycoprotein is an HCV E2 envelope glycoprotein.

In one embodiment of the methods described herein, the cell is present in a subject and the contacting is accomplished by administration of the agent to the subject. Accordingly, the present invention has various applications including the treatment of HCV, as well as the treatment of a subject afflicted with HCV. As used herein, "affected by HCV" means that the subject has at least one cell that has been infected by HCV. As used herein, "treatment" refers to either slowing, halting, or reversing the progression of HCV disease. In the preferred embodiment, "treatment" means the inverse of progression to the point of elimination of the disease As used herein, "treatment" also means the reduction in the number of viral infections, reduction in the number of infectious viral particles, reduction in the number of viral infected cells or the relief of symptoms associated with HCV. Another application of the present invention is to prevent a subject from contracting HCV. As used herein, "HCV donate" refers to an infection with HCV whose genetic information replicates and / or integrates into the host cells.Another application of the present invention is the treatment of a subject infected with HCV, as herein "HCV infection" refers to the introduction of HCV genetic information into a target cell, such as by fusion of the target cell membrane with HCV or an HCV envelope glycoprotein ⁺ cell. The target cell may be a body cell of a subject. In the preferred embodiment, the target cell is a human cell body cell. Another application of the present invention is the inhibition of HCV infection. As used herein, "inhibition of HCV infection" refers to a reduction in the amount of HCV genetic information introduced into a population of target cells compared to the amount that would be introduced without said composition.

What the amount of the compound and / or the agent for administration As far as the subject is concerned, the person skilled in the art will know how to be suitable Quantity is to be determined. As used herein, a dose or amount would be one in sufficient quantities, to inhibit either HCV infection, HCV infection to treat or treat or protect the subject from to be infected with HCV. This amount can be considered an effective Amount to be viewed. The average expert is to able to perform simple titration experiments to determine the amount needed to treat the subject is. The dose of the composition according to the invention depending on the subject and the specific path used Vary in administration. In one embodiment, the dose may be in a range between about 0.1 and about 100,000 μg / kg of body weight lie of the subject. Based on the composition, the Dose continuously, as by continuous pumping, or be supplied at periodic intervals, z. B. to a or several separate occasions. The desired time intervals for multiple Doses of a particular composition can be made by those skilled in the art without undue experimentation be determined.

In one embodiment of the methods described herein, the effective amount of the compound is between about 1 mg and about 50 mg per kg body weight of the subject. In one embodiment, the effective amount of the compound is between about 2 mg and about 40 mg per kg body weight of the subject. In one embodiment, the effective amount of the compound is between about 3 mg and about 30 mg per kg body weight of the subject. In one embodiment, the effective amount of the compound is between about 4 mg and about 20 mg per kg body weight of the subject. In one embodiment, the effective amount of the compound is between about 5 mg and about 10 mg per kg body weight of the subject. The effective amount of the compound may comprise between about 0.000001 mg / kg of body weight and about 100 mg / kg of body weight. In one embodiment, the effective amount may comprise between about 0.001 mg / kg of body weight and about 50 mg / kg of body weight. In another embodiment, the effective amount may range between 0.01 mg / kg body weight and about 10 mg / kg body weight. The effective amount may be based, among other factors, on the size of the compound, the biodegradability of the compound, the bioactivity of the compound, and the bioavailability of the compound. If the compound does not decompose rapidly, is bioavailable, and is highly active, then a lesser amount of potency will be required. The effective amount will be known to those skilled in the art; it will also depend on the size of the compound and the bioactivity of the compound. One skilled in the art is routinely able to perform empirical activity assays for a compound to determine its bioactivity in bioassays and thus the effective amount. In one embodiment of the methods described above, the effective amount of the compound comprises between about 1.0 ng / kg and about 100 mg / kg body weight of the subject. In another embodiment of the methods described above, the effective amount of the compound comprises between about 100 ng / kg and about 50 mg / kg body weight of the subject. In another embodiment of the methods described above, the effective amount of the compound comprises between about 1 μg / kg and about 10 mg / kg body weight of the subject. In a further embodiment of the above The method described comprises the effective amount of the compound between about 100 μg / kg and about 1 mg / kg body weight of the subject.

What as to the time at which the connection and / or the Agent to be administered, the expert is to in the Able to determine when such a connection and / or such Agent should be administered. The administration can be constant over one certain period or periodically and at specific intervals respectively. The connection can be hourly, Every day, weekly, monthly, annually (eg with time-delayed Drug release) or as a single dose. at The administration may be a continuous administration via a act for a specific period, eg B. intravenous administration. In a embodiment The method described herein will become the agent at least once administered daily. In one embodiment of the invention described herein Procedure becomes the agent daily administered. In one embodiment The method described herein becomes the agent on every other Administered day. In one embodiment The method described herein becomes the agent every 6 to 8 days administered. In one embodiment The method described herein becomes the agent weekly administered.

As As used herein, "subject" refers to any animal or an artificial one modified animal capable of being infected by HCV to become. These subjects include, but are not limited to: a human, a primate, an equines, an opines, an avianes, a bovine, porcines, canines, felines or murines Subject. Artificially close modified animals SCID mice with human immune systems, but are not limited to these. The Close animals mice Rats, dogs, guinea pigs, ferrets, rabbits and primates but are not limited to these. In the preferred embodiment is the subject a human? In the subject it can be an "HCV-infected Subject "act, which is a subject in which at least one cell of its own HCV was invaded. In the preferred embodiment, it is in the HCV-infected subject around a human. In the subject it can not be a "not HCV-infected Subject "act, which is a subject in which no own cell of HCV invades has been. In the preferred embodiment the non-HCV infected subject is a human.

As used herein may be "administration" using be carried out by any method known in the art or carried out become. The compound can be administered in several ways be, including, but not limited on, the aerosol, the intravenous, the oral or topical route. The administration may include: intralesional, intraperitoneal, subcutaneous, intramuscular or intravenous injection; Infusion; liposome-mediated administration; topical, intrathecal, gingival pockets, rectal, intrabronchial, nasal, transmucosal, intestinal, oral, ocular or otic administration. In a further embodiment includes administration intrabronchial administration, anal, intrathecal Administration or transdermal administration. The connections and / or agents of the present invention may be administered locally via a capsule which are the time-delayed ones Release of the drug or peptide over a period of time allowed. Compositions with controlled or delayed release of active ingredient shut down the formulation in lipophilic depots (eg fatty acids, waxes, oils). Also encompassed by the invention are polymers (eg with Poloxamers or poloxamines) coated particle compositions, in which the agent is against tissue-specific receptors, ligands or antigen directed antibodies or coupled to ligands of tissue-specific receptors. Further embodiments the compositions of the invention include particulate Forms of protective Coatings, protease inhibitors or permeation enhancers for various Routes of administration, including parenteral, pulmonary, nasal and oral.

at the carrier it can be a diluent, an aerosol, a topical carrier, an aqueous one Solution, a non-aqueous solution or a solid carrier act.

The present invention provides a method of inhibiting HCV infection of cells of a subject susceptible to HCV infection by a method described herein. The present invention provides a method of inhibiting HCV infection of cells of a subject susceptible to HCV infection by a method described herein. The present invention provides a use of a compound for the manufacture of a medicament for preventing one or more cells of a subject from being infected with HCV, the compound being present in an amount sufficient to prevent binding of HCV to DC. Inhibit SIGN and / or DC-SIGNR receptors on the surfaces of the subject's cells and thereby prevent the subject's cell (s) from being infected by HCV. The present invention provides a use of a compound described herein for the manufacture of a medicament in an amount sufficient to promote the binding of HCV To inhibit DC-SIGN or DC-SIGNR receptors on the surface of the cells of the subject. In a preferred embodiment, the subject is a human. in another embodiment, the subject is a SCID BNX mouse (Galun et al., J. Inf. Dis. 172: 25, 1995).

In an embodiment The method described above, the subject before the administration of the Connection to the subject infected with HCV. In one embodiment The method described above does not prevent the subject from Administration of the compound to the subject infected with HCV. In an embodiment In the method described above, the subject is not HCV infected, but was exposed to this.

In an embodiment The methods described herein are for HCV infection receptive Cell to a primary Cell. In one embodiment the cell is a dendritic cell, a placental cell or an endometrial cell. In one embodiment, it is in the cell, a liver cell, a lymph node cell, an endometrial cell in the liver or around a placental cell. In one embodiment The methods described herein are for HCV infection receptive Cell around a eukaryotic cell. In one embodiment of the invention described herein Procedures are those susceptible to HCV infection Cell around a human cell. In one embodiment of the invention described herein Procedures are those susceptible to HCV infection Cell around a mononuclear Cell from peripheral blood. In one embodiment of the invention described herein Procedures are those susceptible to HCV infection Cell around a HeLa cell. In one embodiment of the invention described herein Procedures are those susceptible to HCV infection Cell around a hepatic cell. A hepatic cell can be a HepG2 cell, SK-HEP1 cell, C3A cell or Huh-7 cell lock in, but is not limited to these. in one embodiment the hepatic cell is a primary hepatic cell Cell.

The present invention provides a use of a compound which is an agent described herein or a is described herein composition. In one embodiment the agent or the composition may be sufficient for the viral Reduce stress on a subject. As used herein, "treatment" refers to either slowing down, halting or reversing the progression of HCV disease. In the preferred embodiment "treatment" refers to the reversal of the Progressing to the point of elimination of the disease. As used herein, "treatment" also refers to the reduction the number of viral infections, the reduction in the number of infectious viral Particles, the reduction of the number of virally infected cells or the relief of symptoms associated with HCV. As used herein means "from HCV affected ", the subject has at least one cell associated with HCV is infected.

The The present invention provides a use of one described herein Agent or a composition described herein for the production a drug, wherein the agent or composition be present in a dose which is sufficiently effective to To prevent the subject from contracting HCV.

The The present invention provides a use of one described herein Compound and / or an agent described herein, such as a antibody or a part thereof, a peptide, a polypeptide or oligopeptide, or a nonpeptidyl agent, for the preparation of a pharmaceutical composition for inhibition one for susceptible to HCV infection Cell ready. The present invention provides a use of a Compound described herein and / or one described herein Agent, such as an antibody or a part thereof, a peptide, a polypeptide or oligopeptide, or a non-peptidyl agent, for the preparation of a pharmaceutical A composition for treating HCV infection in a subject ready. The present invention provides a use of one herein described compound and / or an agent described herein, like an antibody or a part thereof, a peptide, a polypeptide or oligopeptide, or a nonpeptidyl agent, for the manufacture of a pharmaceutical Composition for prevention a HCV infection in a subject ready.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGN-Protein, um alle Bindungsstellen für das DC-SIGN-Protein auf dem immobilisierten HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche die Bindung des DC-SIGN-Proteins an das immobilisierte HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGN-Protein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges DC-SIGN-Protein aus dem Komplex verdrängt wird, wobei eine Verdrängung von DC-SIGN-Protein aus dem Komplex anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGN protein to saturate all binding sites for the DC-SIGN protein on the immobilized HCV envelope glycoprotein under conditions involving binding of the DC-SIGN protein allow the immobilized HCV envelope glycoprotein to form a complex;
• c) removing unbound DC-SIGN protein;
• d) contacting the complex with the compound; and
• e) determining whether any DC-SIGN protein is displaced from the complex, wherein displacement of DC-SIGN protein from the complex indicates that the compound binds to the HCV envelope glycoprotein, thereby determining that it is in the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGNR-Protein, um alle Bindungsstellen für das DC-SIGNR-Protein auf dem immobilisierten HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche die Bindung des DC-SIGNR-Proteins an das immobilisierte HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges DC-SIGNR-Protein aus dem Komplex verdrängt wird, wobei eine Verdrängung von DC-SIGNR-Protein aus dem Komplex anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGNR protein to saturate all binding sites for the DC-SIGNR protein on the immobilized HCV envelope glycoprotein under conditions involving binding of the DC-SIGNR protein allow the immobilized HCV envelope glycoprotein to form a complex;
• c) removing unbound DC-SIGNR protein;
• d) contacting the complex with the compound; and
• e) determining whether any DC-SIGNR protein is displaced from the complex, wherein displacement of DC-SIGNR protein from the complex indicates that the compound binds to the HCV envelope glycoprotein, thereby determining that it is in the compound is a compound which is capable of inhibiting HCV infection of the cell.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGN-Proteins mit einer ausreichenden Menge an detektierbarem HCV-Hüllglykoprotein, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGN-Protein unter Bedingungen zu saturieren, welche die Bindung des immobilisierten DC-SIGN-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung von HCV-Hüllglykoprotein aus dem Komplex anzeigt, dass die Verbindung an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the immobilized DC-SIGN protein with a sufficient amount of detectable HCV envelope glycoprotein to saturate all binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGN protein under conditions which inhibit binding of the immobilized DC-SIGN protein. Allow protein to the HCV envelope glycoprotein, so that a complex is formed;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with the compound; and
• e) determining whether any HCV envelope glycoprotein is displaced from the complex, wherein displacement of HCV envelope glycoprotein from the complex indicates that the compound binds to the DC-SIGN protein to thereby determine that the HCV envelope glycoprotein is out of the complex Compound is a compound which is capable of inhibiting a HCV infection of the cell.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGNR-Proteins mit einer ausreichenden Menge an detektierbarem HCV-Hüllglykoprotein, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGNR-Protein unter Bedingungen zu saturieren, welche die Bindung des immobilisierten DC-SIGNR-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit der Verbindung; und
• e) Bestimmung, ob ein beliebiges HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung von HCV-Hüllglykoprotein aus dem Komplex anzeigt, dass die Verbindung an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the immobilized DC-SIGNR protein with a sufficient amount of detectable HCV envelope glycoprotein to saturate all binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGNR protein under conditions which inhibit binding of the immobilized DC-SIGNR protein. Allow protein to the HCV envelope glycoprotein, so that a complex is formed;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with the compound; and
• e) determining whether any HCV envelope glycoprotein is displaced from the complex, wherein displacement of HCV envelope glycoprotein from the complex indicates that the compound binds to the DC-SIGNR protein to thereby determine that the HCV envelope glycoprotein is out of the complex Compound is a compound which is capable of inhibiting a HCV infection of the cell.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGN-Protein, um alle Bindungsstellen für das DC-SIGN-Protein auf dem HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche eine Bindung des DC-SIGN-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGN-Protein;
• c) Messen der Menge an DC-SIGN-Protein, welches an das HCV-Hüllglykoprotein in dem Komplex gebunden wird;
• d) Inkontaktbringen des Komplexes mit der Verbindung, so dass DC-SIGN-Protein aus dem Komplex verdrängt wird;
• e) Messen der Menge an DC-SIGN-Protein, welches in der Anwesenheit der Verbindung an die Verbindung gebunden wird; und
• f) Vergleichen der Menge von DC-SIGN-Protein, welches in Schritt e) an das HCV-Hüllglykoprotein gebunden wurde, mit der in Schritt c) gemessenen Menge, wobei eine reduzierte in Schritt e) gemessene Menge anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGN protein to saturate all binding sites for the DC-SIGN protein on the HCV envelope glycoprotein under conditions involving binding of the DC-SIGN protein to the DCSIGN protein Allow HCV envelope glycoprotein to form a complex;
• b) removing unbound DC-SIGN protein;
• c) measuring the amount of DC-SIGN protein bound to the HCV envelope glycoprotein in the complex;
• d) contacting the complex with the compound such that DC-SIGN protein is displaced from the complex;
• e) measuring the amount of DC-SIGN protein bound to the compound in the presence of the compound; and
• f) comparing the amount of DC-SIGN protein bound to the HCV envelope glycoprotein in step e) with the amount measured in step c), wherein a reduced amount measured in step e) indicates that the compound is attached to the HCV envelope glycoprotein HCV envelope glycoprotein binds to thereby determine that the compound is a compound capable of inhibiting HCV infection of the cell.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit einer ausreichenden Menge an detektierbarem DC-SIGNR-Protein, um alle Bindungsstellen für das DC-SIGNR-Protein auf dem HCV-Hüllglykoprotein unter Bedingungen zu saturieren, welche eine Bindung des DC-SIGNR-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• c) Messen der Menge an DC-SIGNR-Protein, welches an das HCV-Hüllglykoprotein in dem Komplex gebunden wird;
• d) Inkontaktbringen des Komplexes mit der Verbindung, so dass DC-SIGNR-Protein aus dem Komplex verdrängt wird;
• e) Messen der Menge an DC-SIGNR-Protein, welches in der Anwesenheit der Verbindung an die Verbindung gebunden wird; und
• f) Vergleichen der Menge von DC-SIGNR-Protein, welches in Schritt e) an das HCV-Hüllglykoprotein gebunden wurde, mit der in Schritt c) gemessenen Menge, wobei eine reduzierte in Schritt e) gemessene Menge anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein bindet, um dadurch die Verbindung als eine Verbindung zu identifizieren, welche dazu in der Lage ist, eine HCV-Infektion einer Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGNR protein to saturate all binding sites for the DC-SIGNR protein on the HCV envelope glycoprotein under conditions involving binding of the DC-SIGNR protein to the DCSIGNR protein Allow HCV envelope glycoprotein to form a complex;
• b) removing unbound DC-SIGNR protein;
• c) measuring the amount of DC-SIGNR protein bound to the HCV envelope glycoprotein in the complex;
• d) contacting the complex with the compound such that DC-SIGNR protein is displaced from the complex;
• e) measuring the amount of DC-SIGNR protein bound to the compound in the presence of the compound; and
• f) comparing the amount of DC-SIGNR protein bound to the HCV envelope glycoprotein in step e) with the amount measured in step c), wherein a reduced amount measured in step e) indicates that the compound is attached to the HCV envelope glycoprotein HCV envelope glycoprotein binds to thereby identify the compound as a compound capable of inhibiting HCV infection of a cell.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGN-Protein unter Bedingungen, welche die Bindung des DC-SIGN-Proteins an das immobilisierte HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGN-Protein;
• d) Vergleichen der Menge an detektierbarem DC-SIGN-Protein, welche in der Anwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGN-Protein, welche in der Abwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an DC-SIGN-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with the compound and detectable DC-SIGN protein under conditions permitting binding of the DC-SIGN protein to the immobilized HCV envelope glycoprotein to form a complex;
• c) removing unbound DC-SIGN protein;
• d) comparing the amount of detectable DC-SIGN protein bound in the presence of the compound to the immobilized HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGN protein immobilized in the absence of the compound HCV envelope glycoprotein binds;
• e) wherein a reduced amount of DC-SIGN protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGN protein to thereby determine that the compound is a A compound which is capable of inhibiting a HCV infection of the cell.

In an embodiment The method described herein is the amount of detectable DC-SIGN sufficient to bind all the DC-SIGN protein binding sites on the HCV envelope glycoprotein to saturate.

• a) Immobilisieren eines HCV-Hüllglykoproteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGNR-Protein unter Bedingungen, welche die Bindung des DC-SIGNR-Proteins an das immobilisierte HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• d) Vergleichen der Menge an detektierbarem DC-SIGNR-Protein, welche in der Anwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGNR-Protein, welche in der Abwesenheit der Verbindung an das immobilisierte HCV-Hüllglykoprotein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an DC-SIGNR-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing an HCV envelope glycoprotein on a solid support;
• b) contacting the immobilized HCV envelope glycoprotein with the compound and detectable DC-SIGNR protein under conditions permitting binding of the DC-SIGNR protein to the immobilized HCV envelope glycoprotein to form a complex;
• c) removing unbound DC-SIGNR protein;
• d) comparing the amount of detectable DC-SIGNR protein bound in the presence of the compound to the immobilized HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGNR protein immobilized in the absence of the compound HCV envelope glycoprotein binds;
• e) wherein a reduced amount of DC-SIGNR protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that the compound is a Is a compound capable of doing so To inhibit HCV infection of the cell.

In an embodiment The method described herein is the amount of detectable DC-SIGNR sufficient to detect all binding sites for the DC-SIGNR protein on the HCV envelope glycoprotein to saturate.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGN-Proteins mit der Verbindung und detektierbarem HCV-Hüllglykoprotein unter Bedingungen, welche die Bindung des immobilisierten DC-SIGN-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Vergleichen der Menge an detektierbarem HCV-Hüllglykoprotein, welche in der Anwesenheit der Verbindung an das immobilisierte DC-SIGN-Protein in dem Komplex gebunden wird mit der Menge an detektierbarem HCV-Hüllglykoprotein, welche in der Abwesenheit der Verbindung an das immobilisierte DC-SIGN-Protein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an HCV-Hüllglykoprotein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the immobilized DC-SIGN protein with the compound and detectable HCV envelope glycoprotein under conditions permitting binding of the immobilized DC-SIGN protein to the HCV envelope glycoprotein to form a complex;
• c) removing unbound HCV envelope glycoprotein;
• d) comparing the amount of detectable HCV envelope glycoprotein bound in the complex in the presence of the compound to the immobilized DC-SIGN protein in the complex with the amount of detectable HCV envelope glycoprotein which in the absence of the compound binds to the immobilized DC SIGN protein binds;
• e) wherein a reduced amount of HCV envelope glycoprotein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGN protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

In an embodiment The method described herein is the amount of detectable HCV envelope glycoprotein sufficient to detect all binding sites for the HCV envelope glycoprotein on the DC-SIGN protein to saturate.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGNR-Proteins mit der Verbindung und detektierbarem HCV-Hüllglykoprotein unter Bedingungen, welche die Bindung des immobilisierten DC-SIGNR-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Vergleichen der Menge an detektierbarem HCV-Hüllglykoprotein; welche in der Anwesenheit der Verbindung an das immobilisierte DC-SIGNR-Protein in dem Komplex gebunden wird mit der Menge an detektierbarem HCV-Hüllglykoprotein, welche in der Abwesenheit der Verbindung an das immobilisierte DC-SIGNR-Protein bindet;
• e) wobei eine reduzierte in Anwesenheit der Verbindung gemessene Menge an HCV-Hüllglykoprotein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the immobilized DC-SIGNR protein with the compound and detectable HCV envelope glycoprotein under conditions permitting binding of the immobilized DC-SIGNR protein to the HCV envelope glycoprotein to form a complex;
• c) removing unbound HCV envelope glycoprotein;
• d) comparing the amount of detectable HCV envelope glycoprotein; which, in the presence of the compound, binds to the immobilized DC-SIGNR protein in the complex with the amount of detectable HCV envelope glycoprotein which, in the absence of the compound, binds to the immobilized DC-SIGNR protein;
• e) wherein a reduced amount of HCV envelope glycoprotein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

In an embodiment The method described herein is the amount of detectable HCV envelope glycoprotein sufficient to all binding sites for the HCV envelope glycoprotein on the DC-SIGNR protein to saturate.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGN-Protein unter Bedingungen, welche die Bindung des DC-SIGN-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGN-Protein;
• c) Vergleichen der Menge an detektierbarem DC-SIGN-Protein, welches in der Anwesenheit der Verbindung an das HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGN- Protein, welches in der Abwesenheit der Verbindung an die Verbindung bindet; wobei eine reduzierte in der Anwesenheit der Verbindung gemessene Menge an DC-SIGN-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGN-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention discloses a method of determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with the compound and detectable DC-SIGN protein under conditions permitting binding of the DC-SIGN protein to the HCV envelope glycoprotein to form a complex;
• b) removing unbound DC-SIGN protein;
• c) comparing the amount of detectable DC-SIGN protein bound in the presence of the compound to the HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGN protein which binds to the compound in the absence of the compound ; wherein a reduced amount of DC-SIGN protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGN protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

In an embodiment The method described herein is the amount of detectable DC-SIGN protein sufficient to bind all the DC-SIGN protein binding sites on the HCV envelope glycoprotein to saturate.

• a) Inkontaktbringen eines HCV-Hüllglykoproteins mit der Verbindung und detektierbarem DC-SIGNR-Protein unter Bedingungen, welche die Bindung des DC-SIGNR-Proteins an das HCV-Hüllglykoprotein zulassen, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem DC-SIGNR-Protein;
• c) Vergleichen der Menge an detektierbarem DC-SIGNR-Protein, welches in der Anwesenheit der Verbindung an das HCV-Hüllglykoprotein in dem Komplex gebunden wird mit der Menge an detektierbarem DC-SIGNR-Protein, welches in der Abwesenheit der Verbindung an die Verbindung bindet; wobei eine reduzierte in der Anwesenheit der Verbindung gemessene Menge an DC-SIGNR-Protein anzeigt, dass die Verbindung an das HCV-Hüllglykoprotein oder an das DC-SIGNR-Protein bindet, um dadurch zu bestimmen, dass es sich bei der Verbindung um eine Verbindung handelt, welche dazu in der Lage ist, eine HCV-Infektion der Zelle zu inhibieren.

The present invention provides a method for determining whether a compound is capable of inhibiting HCV infection of a cell, comprising:

• a) contacting an HCV envelope glycoprotein with the compound and detectable DC-SIGNR protein under conditions permitting binding of the DC-SIGNR protein to the HCV envelope glycoprotein to form a complex;
• b) removing unbound DC-SIGNR protein;
• c) comparing the amount of detectable DC-SIGNR protein bound in the presence of the compound to the HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGNR protein which binds to the compound in the absence of the compound ; wherein a reduced amount of DC-SIGNR protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that the compound is a compound which is capable of inhibiting HCV infection of the cell.

In an embodiment The method described herein is the amount of detectable DC-SIGNR protein sufficient to all binding sites for the DC-SIGNR protein on the HCV envelope glycoprotein too saturate.

In The method described herein may make a unit detectable thereby be made marked with a detectable marker becomes. So z. In one embodiment of the invention described herein Method the detectable DC-SIGN protein with a detectable Markers marked. In one embodiment The method described herein involves the detectable DC-SIGNR protein labeled a detectable marker. In one embodiment The method described herein becomes the detectable HCV envelope glycoprotein detected with a detectable marker. The skilled person is various Types of detectable markers known. Such detectable Close the marker radioactive, colorimetric, luminescent and fluorescent Markers, but are not limited to these.

• a) Immobilisieren von einem oder beiden der HCV-Hüllglykoprotein auf einem festen Träger;
• b) Inkontaktbringen des Resultats aus Schritt a) mit dem Agens;
• c) Inkontaktbringen des Resultats aus Schritt c) mit einer detektierbaren Form von DC-SIGN-Protein unter Bedingungen, welche die Bindung des detektierbaren DC-SIGN-Proteins in der Abwesenheit der Verbindung zulassen;
• d) Detektieren der Menge an gebundenem detektierbaren DC-SIGN-Protein, wobei eine Reduktion der Menge an gebundenem DC-SIGN-Protein im Vergleich zu einer in der Abwesenheit des Agens gebundenen Menge somit das Agens als eines identifiziert, welches die Bindung von HCV an das DC-SIGN inhibiert.

The present invention discloses a method for identifying an agent that inhibits the binding of HCV to DC-SIGN, comprising:

• a) immobilizing one or both of the HCV envelope glycoprotein on a solid support;
• b) contacting the result of step a) with the agent;
• c) contacting the result of step c) with a detectable form of DC-SIGN protein under conditions which allow binding of the detectable DC-SIGN protein in the absence of the compound;
• d) detecting the amount of bound detectable DC-SIGN protein, wherein a reduction in the amount of bound DC-SIGN protein compared to an amount bound in the absence of the agent thus identifies the agent as one which inhibits the binding of HCV the DC-SIGN inhibited.

• a) Immobilisieren von einem oder beiden der HCV-Hüllglykoprotein auf einem festen Träger;
• b) Inkontaktbringen des Resultats aus Schritt a) mit dem Agens;
• c) Inkontaktbringen des Resultats aus Schritt c) mit einer detektierbaren Form von DC-SIGNR-Protein unter Bedingungen, welche die Bindung des detektierbaren DC-SIGNR-Proteins in der Abwesenheit der Verbindung zulassen;
• d) Detektieren der Menge an gebundenem detektierbaren DC-SIGNR-Protein, wobei eine Reduktion der Menge an gebundenem DC-SIGNR-Protein im Vergleich zu einer in der Abwesenheit des Agens gebundenen Menge somit das Agens als eines identifiziert, welches die Bindung von HCV an das DC-SIGNR inhibiert.

The present invention discloses a method for identifying an agent that inhibits the binding of HCV to DC-SIGNR, comprising:

• a) immobilizing one or both of the HCV envelope glycoprotein on a solid support;
• b) contacting the result of step a) with the agent;
• c) contacting the result of step c) with a detectable form of DC-SIGNR protein under conditions which allow the binding of the detectable DC-SIGNR protein in the absence of the compound;
• d) detecting the amount of bound detectable DC-SIGNR protein, wherein a reduction in the amount of bound DC-SIGNR protein compared to an amount bound in the absence of the agent thus identifies the agent as one which inhibits the binding of HCV the DC-SIGNR inhibits.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des Resultats aus Schritt a) mit dem Agens;
• c) Inkontaktbringen des Resultats aus Schritt b) mit einer detektierbaren Form von einem oder mehreren des HCV-Hüllglykoproteins unter Bedingungen, welche die Bindung des detektierbaren HCV-Hüllglykoproteins in der Abwesenheit der Verbindung zulassen;
• d) Detektieren der Menge an gebundenem(n) detektierbaren HCV-Hüllglykoprotein(en), wobei eine Reduktion der Menge an gebundenem(n) HCV-Hüllglykoprotein(en) im Vergleich zu einer in der Abwesenheit des Agens gebundenen Menge somit das Agens als eines identifiziert, welches die Bindung von HCV an das DC-SIGN inhibiert.

The present invention discloses a method for identifying an agent that inhibits the binding of HCV to DC-SIGN, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the result of step a) with the agent;
• c) contacting the result of step b) with a detectable form of one or more of the HCV envelope glycoprotein under conditions involving binding of the detectable HCV envelope glycopro in the absence of the connection;
• d) detecting the amount of bound detectable HCV envelope glycoprotein (s), wherein a reduction in the amount of bound HCV envelope glycoprotein (s) thus compared to an amount bound in the absence of the agent, constitutes the agent as one which inhibits the binding of HCV to DC-SIGN.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des Resultats aus Schritt a) mit dem Agens;
• c) Inkontaktbringen des Resultats aus Schritt c) mit einer detektierbaren Form von einem oder mehreren des HCV-Hüllglykoproteins unter Bedingungen, welche die Bindung des detektierbaren HCV-Hüllglykoproteins in der Abwesenheit der Verbindung zulassen;
• d) Detektieren der Menge an gebundenem(n) detektierbaren HCV-Hüllglykoprotein(en), wobei eine Reduktion der Menge an gebundenem(n) HCV-Hüllglykoprotein(en) im Vergleich zu einer in der Abwesenheit des Agens gebundenen Menge somit das Agens als eines identifiziert, welches die Bindung von HCV an das DC-SIGNR inhibiert.

The present invention discloses a method for identifying an agent that inhibits the binding of HCV to DC-SIGNR, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the result of step a) with the agent;
• c) contacting the result of step c) with a detectable form of one or more of the HCV envelope glycoprotein under conditions allowing binding of the detectable HCV envelope glycoprotein in the absence of the compound;
• d) detecting the amount of bound detectable HCV envelope glycoprotein (s), wherein a reduction in the amount of bound HCV envelope glycoprotein (s) thus compared to an amount bound in the absence of the agent, constitutes the agent as one which inhibits the binding of HCV to DC-SIGNR.

In one embodiment of the methods described herein, the solid support is a well of a microtiter plate. In another embodiment, the solid support is a bead. In another embodiment, the solid support is a surface plasmon resonance sensor chip. The surface plasmon resonance sensor chip may include preimmobilized streptavidin. In one embodiment, the surface plasmon resonance sensor chip is a BIAcore ^™ chip.

In an embodiment The method described above is the detectable molecule with a detectable Markers marked. In a further embodiment of the previously described Method, the detectable molecule is detected by passing it with another compound in contact, which both to Binding of the detectable molecule capable as well as detectable. The detectable markers shut down the previously described one.

As used herein the terms "agent" and "compound" mean both protein and as well as non-protein residues. In one embodiment, it is at the agent / compound around a small molecule. In a another embodiment the agent / compound is a protein. In which Protein may be, for. B. be an antibody, which against a part of an HCV envelope glycoprotein is directed. The agent / compound may be from a library of low molecular weight compounds or a library derived from extracts of plants or other organisms. In one embodiment the agent is known. In a separate embodiment, the agent / s Connection not known in advance. The agents / compounds of to close the present invention but are not limited on: compounds or molecular entities such as peptides, polypeptides and other organic or inorganic molecules, and combinations thereof.

Compounds of the invention inhibit HCV infection of cells susceptible to HCV infection. The compounds of the invention preferably have a specificity for prevention or inhibition of infection by HCV and inhibit none Infection by other viruses, such as HIV, which is DC-SIGN or Use DC-SIGNR for infection. In addition, affect or inhibiting the compounds of the invention members the immunoglobulin superfamily not, especially impair the compounds ICAM-2 or ICAM-3 or ICAM-2-like or ICAM-3-like Not molecules.

As used herein, the terms "agent" and "compound" are interchangeable. In one embodiment of the methods described herein, the agent is an antibody or part of an antibody. In one embodiment of the antibody, the antibody is a monoclonal antibody. In one embodiment of the antibody, the antibody is a polyclonal antibody. In one embodiment of the antibody, the antibody is a humanized antibody. In one embodiment of the antibody, the antibody is a chimeric antibody. The part of the antibody may comprise a light chain of the antibody. The part of the antibody may comprise a heavy chain of the antibody. The part of the antibody may comprise a Fab portion of the antibody. The portion of the antibody may comprise an F (ab ') ₂ portion of the antibody. The portion of the antibody may comprise an Fd portion of the antibody. The portion of the antibody may comprise a Fv portion of the antibody. The part of the antibody may comprise a variable domain of the antibody. The portion of the antibody may comprise one or more CDR domains of the antibody.

In an embodiment The method described herein is the agent to a polypeptide. In one embodiment The method described herein is the agent to an oligopeptide. In one embodiment of the invention described herein The method is the agent is a non-peptidyl agent. In one embodiment when the non-peptidyl agent is a compound with a molecular weight of less than 500 daltons.

• a) Identifikation einer Verbindung, welche eine HCV-Infektion einer Zelle gemäß einem hierin beschriebenen Verfahren inhibiert;
• b) Vermischen der auf diese Weise identifizierten Verbindung oder eines Homologs oder eines Derivats davon mit einem Träger, um dadurch eine Zusammensetzung zu erhalten.

The present invention discloses a process for obtaining a composition comprising:

• a) identification of a compound which inhibits HCV infection of a cell according to a method described herein;
• b) mixing the thus identified compound or a homologue or a derivative thereof with a carrier to thereby obtain a composition.

• a) Identifikation einer Verbindung, welche die Bindung von HCV an DC-SIGN gemäß einem der zuvor beschriebenen Verfahren inhibiert; und
• b) Vermischen der auf diese Weise identifizierten Verbindung oder eines Homologs oder eines Derivats davon mit einem Träger.

The present invention discloses a process for obtaining a composition comprising:

• a) identification of a compound which inhibits the binding of HCV to DC-SIGN according to one of the methods described above; and
• b) mixing the compound identified in this way or a homologue or a derivative thereof with a carrier.

• a) Identifikation einer Verbindung, welche die Bindung von HCV an DC-SIGNR gemäß einem der zuvor beschriebenen Verfahren inhibiert; und
• b) Vermischen der auf diese Weise identifizierten Verbindung oder eines Homologs oder eines Derivats davon mit einem Träger.

The present invention discloses a process for obtaining a composition comprising:

• a) identification of a compound which inhibits the binding of HCV to DC-SIGNR according to one of the methods described above; and
• b) mixing the compound identified in this way or a homologue or a derivative thereof with a carrier.

In an embodiment This method for obtaining a composition comprises said method further, obtaining the identified compound before she with the carrier is mixed.

The The present invention discloses a method of treatment or prevention liver disease in a subject comprising administration an effective amount of a compound capable of doing so is the binding of an HCV envelope glycoprotein to one on the surface to inhibit cells of the subject present DC-SIGN protein, to the subject, thereby causing liver disease in a subject to treat or prevent. The present invention provides a method of treating or preventing liver disease in a subject ready, comprising administering an effective Amount of a compound capable of binding an HCV envelope glycoprotein to one on the surface to inhibit cells of the subject existing DC-SIGNR protein, to the subject, thereby causing liver disease in a subject to treat or prevent. In one embodiment of the invention described herein Procedure is the liver disease to hepatitis. In one embodiment The method described herein is liver disease around cirrhosis.

The The present invention discloses a method of treatment or prevention of a hepatocellular Carcinoma in a subject comprising the administration of an effective Amount of a compound capable of binding an HCV envelope glycoprotein to one on the surface to inhibit cells of the subject present DC-SIGN protein, to the subject, thereby affecting the hepatocellular carcinoma in a subject to treat or avoid. The present invention discloses a method of treating or preventing hepatocellular carcinoma in a subject comprising administering an effective amount a compound which is capable of binding a HCV envelope glycoprotein to one on the surface to inhibit cells of the subject present DC-SIGNR protein the subject, thereby affecting the hepatocellular carcinoma in a subject to treat or avoid.

• a) Immobilisieren eines DC-SIGN-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGN-Proteins mit einer Menge an detektierbarem HCV-Hüllglykoprotein, welche ausreichend ist, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGN-Protein zu saturieren, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• e) Detektieren, ob HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung des HCV-Hüllglykoproteins aus dem Komplex die Anwesenheit von in der Probe vorhandenen anti-HCV-Antikörpern anzeigt, um dadurch eine HCV-Infektion des Subjekts zu diagnostizieren.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) immobilizing a DC-SIGN protein on a solid support;
• b) contacting the immobilized DC-SIGN protein with an amount of detectable HCV envelope glycoprotein sufficient to saturate all binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGN protein to form a complex;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with a suitable sample obtained from the subject;
• e) detecting whether HCV envelope glycoprotein is displaced from the complex, wherein displacement of the HCV envelope glycoprotein from the complex indicates the presence of anti-HCV antibodies present in the sample to thereby diagnose HCV infection of the subject.

• a) Immobilisieren eines DC-SIGNR-Proteins auf einem festen Träger;
• b) Inkontaktbringen des immobilisierten DC-SIGNR-Proteins mit einer Menge an detektierbarem HCV-Hüllglykoprotein, welche ausreichend ist, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem immobilisierten DC-SIGNR-Protein zu saturieren, so dass ein Komplex gebildet wird;
• c) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• d) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• e) Detektieren, ob HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung des HCV-Hüllglykoproteins aus dem Komplex die Anwesenheit von in der Probe vorhandenen anti-HCV-Antikörpern anzeigt, um dadurch eine HCV-Infektion des Subjekts zu diagnostizieren.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) immobilizing a DC-SIGNR protein on a solid support;
• b) contacting the immobilized DC-SIGNR protein with an amount of detectable HCV envelope glycoprotein sufficient to saturate all binding sites for the HCV envelope glycoprotein on the immobilized DC-SIGNR protein to form a complex;
• c) removing unbound HCV envelope glycoprotein;
• d) contacting the complex with a suitable sample obtained from the subject;
• e) detecting whether HCV envelope glycoprotein is displaced from the complex, wherein displacement of the HCV envelope glycoprotein from the complex indicates the presence of anti-HCV antibodies present in the sample to thereby diagnose HCV infection of the subject.

• a) Inkontaktbringen von DC-SIGN-Protein mit ausreichend HCV-Hüllglykoprotein, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem DC-SIGN-Protein zu saturieren, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• c) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• d) Detektieren, ob HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung des HCV-Hüllglykoproteins aus dem Komplex die Anwesenheit von in der Probe vorhandenen anti-HCV-Antikörpern anzeigt, um dadurch eine HCV-Infektion des Subjekts zu diagnostizieren.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) contacting DC-SIGN protein with sufficient HCV envelope glycoprotein to saturate all binding sites for the HCV envelope glycoprotein on the DC-SIGN protein to form a complex;
• b) removing unbound HCV envelope glycoprotein;
• c) contacting the complex with a suitable sample obtained from the subject;
• d) detecting whether HCV envelope glycoprotein is displaced from the complex, wherein displacement of the HCV envelope glycoprotein from the complex indicates the presence of anti-HCV antibodies present in the sample to thereby diagnose HCV infection of the subject.

• a) Inkontaktbringen von DC-SIGNR-Protein mit ausreichend HCV-Hüllglykoprotein, um alle Bindungsstellen für das HCV-Hüllglykoprotein auf dem DC-SIGNR-Protein zu saturieren, so dass ein Komplex gebildet wird;
• b) Entfernen von nicht gebundenem HCV-Hüllglykoprotein;
• c) Inkontaktbringen des Komplexes mit einer geeigneten von dem Subjekt erhaltenen Probe;
• d) Detektieren, ob HCV-Hüllglykoprotein aus dem Komplex verdrängt wird, wobei eine Verdrängung des HCV-Hüllglykoproteins aus dem Komplex die Anwesenheit von in der Probe vorhandenen anti-HCV-Antikörpern anzeigt, um dadurch eine HCV-Infektion des Subjekts zu diagnostizieren.

The present invention discloses a method for diagnosing HCV infection of a subject, comprising:

• a) contacting DC-SIGNR protein with sufficient HCV envelope glycoprotein to saturate all binding sites for the HCV envelope glycoprotein on the DC-SIGNR protein to form a complex;
• b) removing unbound HCV envelope glycoprotein;
• c) contacting the complex with a suitable sample obtained from the subject;
• d) detecting whether HCV envelope glycoprotein is displaced from the complex, wherein displacement of the HCV envelope glycoprotein from the complex indicates the presence of anti-HCV antibodies present in the sample to thereby diagnose HCV infection of the subject.

The ability a DC-SIGN protein, a DC-SIGNR protein or a functional equivalent of binding HCV allows the use of the protein as a diagnostic for an HCV infection, e.g. B. in an ELISA (enzyme linked immunosorbent assay). In one embodiment could a soluble one Form of a DC-SIGN-protein and / or a DC-SIGNR-protein used, serum antibodies to detect HCV. In a preferred embodiment is the DC-SIGN protein and / or the DC-SIGNR protein or functional equivalent of which on a solid support immobilized and in contact with the HCV envelope glycoprotein (s) which is an E1-HCV envelope glycoprotein, an E2-HCV envelope glycoprotein or both can act. The contacting can be in the presence or in the absence of serum or serum antibodies. In an assay of this form leads a competitive binding between antibodies and the HCV envelope glycoprotein (s) in particular to binding to its immobilized protein, that the bound HCV protein as a measure of antibodies in the serum sample is used. The amount of bound HCV envelope glycoprotein (s) is then detected. The HCV envelope glycoprotein (s) can / can with radioactive, enzymatic, biotin, fluorescent or other detectable markers are labeled to the detection to enable.

The The present invention discloses methods for diagnosing HCV infection in a subject using a method known to those skilled in the art, including, but not limited on, a sandwich assay and a competition assay.

• 1) Erhalt einer geeigneten Probe von DC-SIGN- und/oder DC-SIGNR-Protein;
• 2) Inkontaktbringen des DC-SIGN- und/oder DC-SIGNR-Proteins mit einem HCV-Hüllglykoprotein, so dass ein Komplex gebildet wird;
• 3) Erhalt einer geeigneten Probe von dem Subjekt und Inkontaktbringen des HCV-Hüllglykoproteins mit der Probe unter Bedingungen, welche die Bildung eines Komplexes zwischen dem HCV-Hüllglykoprotein und beliebigen in der Probe des Subjekts anwesenden anti-HCV-Hüllglykoprotein-Antikörpern zulassen;
• 4) Inkontaktbringen der gebundenen anti-HCV-Hüllglykoprotein-Antikörper mit detektierbaren anti-human-IgG-Antikörpern welche an beliebige gebundene anti-HCV-Hüllglykoprotein-Antikörper binden würden;
• 5) Detektieren der anti-human-IgG-Antikörper, wobei die Anwesenheit solcher Antikörper anzeigt, dass das Subjekt mit HCV infiziert ist.

So is z. For example, an embodiment of a sandwich assay is as follows:

• 1) Obtain a suitable sample of DC-SIGN and / or DC-SIGNR protein;
• 2) contacting the DC-SIGN and / or DC-SIGNR protein with an HCV envelope glycoprotein to form a complex;
• 3) obtaining a suitable sample from the subject and contacting the HCV envelope glycoprotein with the sample under conditions which permit formation of a complex between the HCV envelope glycoprotein and any anti-HCV envelope glycoprotein antibodies present in the subject's sample;
• 4) contacting the bound anti-HCV envelope glycoprotein antibodies with detectable anti-human IgG antibodies which would bind to any bound anti-HCV envelope glycoprotein antibodies;
• 5) detecting the anti-human IgG antibodies, wherein the presence of such antibodies indicates that the subject is infected with HCV.

• 1) Erhalt einer geeigneten Probe von DC-SIGN- und/oder DC-SIGNR-Protein;
• 2) Inkontaktbringen des DC-SIGN- und/oder DC-SIGNR-Proteins mit einem HCV-Hüllglykoprotein, so dass ein Komplex gebildet wird;
• 3) Inkontaktbringen des HCV-Hüllglykoproteins mit einer Probe des Subjekts unter Bedingungen, welche die Bindung zwischen beliebigen in der Probe anwesenden anti-HCV-Hüllglykoprotein-Antikörpern und dem HCV-Hüllglykoprotein zulassen;
• 4) ebenfalls Inkontaktbringen des HCV-Hüllglykoproteins mit detektierbaren anti-HCV-Hüllglykoprotein-Antikörpern unter Bedingungen, welche eine Bindung zwischen den detektierbaren anti-HCV-Hüllglykoprotein-Antikörpern und dem HCV-Hüllglykoprotein zulassen;
• 5) Bestimmen der Menge an detektierbaren gebundenen anti-HCV-Hüllglykoprotein-Antikörpern im Vergleich zu der in der Abwesenheit einer beliebigen Probe des Subjekts gebunden Menge, wobei eine erhöhte in der Abwesenheit der Probe gemessene Menge anzeigt, dass das Subjekt mit HCV infiziert ist.

So is z. For example, one embodiment of a competition assay is as follows:

• 1) Obtain a suitable sample of DC-SIGN and / or DC-SIGNR protein;
• 2) contacting the DC-SIGN and / or DC-SIGNR protein with an HCV envelope glycoprotein to form a complex;
• 3) contacting the HCV envelope glycoprotein with a sample of the subject under conditions permitting binding between any anti-HCV envelope glycoprotein antibodies present in the sample and the HCV envelope glycoprotein;
• 4) also contacting the HCV envelope glycoprotein with detectable anti-HCV envelope glycoprotein antibodies under conditions which allow binding between the detectable anti-HCV envelope glycoprotein antibodies and the HCV envelope glycoprotein;
• 5) Determining the amount of detectable bound anti-HCV envelope glycoprotein antibodies as compared to the amount bound in the absence of any sample of the subject, wherein an increased amount measured in the absence of the sample indicates that the subject is infected with HCV.

In an embodiment The methods and assays described herein are the sample of the subject around a serum sample. In one embodiment the DC-SIGN and / or DC-SIGNR protein is immobilized. The before described method can Include washes, to wash out unbound components, including, however not limited on unbound HCV envelope glycoprotein, unbound sample of the subject, unbound anti-HCV envelope glycoprotein antibodies as well as not bound detectable anti-human IgG antibodies. In one embodiment are the detectable anti-human IgG antibodies with a detectable Markers marked. In one embodiment are the detectable anti-HCV envelope glycoprotein antibodies with labeled a detectable marker. In one embodiment the amount of anti-human IgG antibodies detected is compared to an amount which was measured in the absence of HCV envelope glycoprotein, so as to obtain a basic reading.

The The present invention discloses an article of manufacture comprising a solid carrier with an agent attached to it operatively attached thereto Location is, in a specific way, a complex with a on an HCV envelope glycoprotein existing domain to build.

at the solid support It may be any known in the art carrier act on which the agent can be surgically attached. firm carrier shut down z. Natural or synthetic polymers. Synthetic polymers include, for. B. Polystyrene, polyethylene and polypropylene. Natural polymers shut down z. B. latex. The solid carrier can z. B. selected be from the group consisting of a bead, a vessel and a Filter. The use of solid carriers in the form of beads is widely used and these are for the expert readily available. Close beads z. As latex and polystyrene beads.

at the vessel can be any vessel, in which a body fluid is kept or with which such a liquid comes into contact. The vessel may, for. B. in the form of a bag or hose. In the preferred embodiment If the vessel is a Bag which is specific for the uptake and / or storage of blood or blood components is provided.

Of the Use of solid straps in the form of filters is widely used and these are for the expert readily available. Close the filter z. B. polyester filters (e.g., polyester leucofiltration devices) and cellulose acetate filters.

The agent attached to the solid support may be either a protein or a nonprotein agent. In one embodiment, the agent is DC-SIGN and / or DC SIGNR. In one embodiment, the agent is an antibody or a portion thereof. Such an antibody may be an antibody capable of binding to an HCV envelope glycoprotein.

As used herein means "operational attached "attached in a way that the formation of a complex between the attached agent and that present on an HCV envelope glycoprotein domain allowed. Method for the surgical fixation of an agent a solid carrier are well known to those skilled in the art.

As used herein means "to to be able to create a complex in a specific way with one on an HCV envelope glycoprotein existing domain to form "to it to be able to form a complex with one on an HCV envelope glycoprotein existing domain, but not with any other domain, form.

In an embodiment it is the one present on the HCV envelope glycoprotein Domain around a conserved domain. As used herein, a "conserved domain" refers to an envelope glycoprotein domain which on at least 90% of all strains from HCV in unchanged Structure is present. In the preferred embodiment, it is at the on the HCV envelope glycoprotein existing domain around the DC-SIGN and / or DC-SIGNR binding domain of the HCV envelope glycoprotein. In a further embodiment it is the one present on the HCV envelope glycoprotein domain around an unpreserved domain.

The The present invention further discloses an article of manufacture comprising a solid support, to which a plurality of agents are operatively attached, from which each one is capable of, in a specific way a complex with an HCV envelope glycoprotein present domain to build.

As As used herein, a "variety of agents" refers to at least two Agents. In one embodiment The variety of agents consists of a variety of DC-SIGN and / or DC-SIGNR based molecules. In a further embodiment the multitude of agents consists of a multitude of antibodies. In a further embodiment For example, the plurality of agents include one antibody and one on DC-SIGN and / or DC-SIGNR based molecule.

The The present invention further provides a water-soluble Agent which (a) is capable of specific A complex with a complex on a HCV envelope glycoprotein existing domain and (b) comprises a residue capable of doing so is, in a specific way, a complex with a known one Ligand, said residue being the removal of the agent from a sample by contacting it with an immobilized one Form of the known ligand allowed. As used herein, "water-soluble" means capable of to be in water at 4 ° C in soluble form in a concentration of at least 1 pM.

The Use of a remainder which is capable of specific ones Way to form a complex with a known ligand, is commonly referred to in the art as "molecular labeling" Rest can z. B. selected be from the group consisting of a small molecule and a Protein. The ligand closes z. A metal ion, a small molecule, a peptide or a protein but is not limited to these. Specific examples for combinations close rest / ligand but are not limited on: (a) oligohistidine / nickel ion, (b) glutathione S-transferase / glutathione, (c) biotin / streptavidin; and (d) the HA peptide YPYDVPDYA / anti-HA peptide antibody. Of the The remainder can be attached by any means known to those skilled in the art be like z. B. on chemical or genetic.

The The present invention further discloses a method of treatment a body fluid sample, to make it HCV or HCV envelope glycoprotein if present in the sample, including contacting the sample under suitable conditions an article of manufacture comprising a solid support which is operably attached to an agent capable of doing so is, in a specific way, a complex with one on one HCV envelope glycoprotein existing domain to form, resulting in HCV or HCV envelope glycoprotein is removed from the sample, if present in the sample was.

As used herein, "treatment of a body fluid sample to remove HCV therefrom" means either (a) to deprive the HCV in the body fluid sample of the ability to intrude into target cells (b) to physically separate HCV from the sample of body fluid, or (c) a combination of (a) and (b), among which Condition that the fraction of HCV present in the resulting sample and capable of invasion of target cells does not exceed 50% of the amount of HCV present prior to the removal of HCV in the sample. As used herein, a target cell includes a cell having DC-SIGN and / or DC-SIGNR present on its surface, wherein the DC-SIGN and / or DC-SIGNR expressing cell is capable of being specifically linked to it in Contact brought HCV to merge and fuse with it.

at suitable conditions for contacting the sample with the article of manufacture according to the invention These are conditions that define the formation of a complex between the agent and HCV. Such conditions are known to the skilled person.

The The present invention further discloses a method of treatment a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising contacting the sample with an appropriate amount a water-soluble Agent that is capable of doing so in a specific way a complex with an HCV envelope glycoprotein present domain to form, so that a complex between the agent and HCV, provided present in the sample, formed and thereby the probability is reduced that a subject as a result of contact with the Sample is infected with HCV.

The The present invention discloses a method for substantial reduction the amount of HCV envelope glycoprotein in a body fluid sample, comprising contacting the sample with an appropriate amount a water-soluble Agens, which is capable of, in a specific way a Complex with an HCV envelope glycoprotein present domain to form, so that a complex between the agent and HCV, provided present in the sample, and thereby the amount of HCV envelope glycoprotein is reduced in the sample.

In an embodiment the blood of HCV-infected individuals is passed through filters, on which DC-SIGN and / or DC-SIGNR based proteins or antibody were immobilized. This would the removal of HCV virions and / or HCV envelope glycoprotein from the blood allow. The presence of HCV envelope glycoprotein in the blood can z. By binding to DC-SIGN and / or DC-SIGNR expressing cells and inhibiting the immune response or by the initiation of apoptosis of these cells may be pathogenic.

In the preferred embodiment is the subject a human? As used herein means "the Probability that a subject becomes infected with HCV, essential to reduce "the Probability that a subject becomes infected with HCV to reduce at least twice. Lies with a subject z. For example, there is a 1% chance that it will be infected with HCV will, so would a twofold reduction in the probability that the subject infected with HCV will cause that the subject has a 0.5% probability that it becomes infected with HCV. In one embodiment, the probability of that a subject becomes infected with HCV, significantly reducing to reduce this probability by at least tenfold. In the preferred embodiment means the likelihood that a subject becomes infected with HCV will significantly reduce this probability by at least to reduce the hundredfold.

As used herein means that "the subject is infected with HCV becomes "the invasion the subject's own cells through HCV.

As As used herein, "contact with a body fluid sample" means any contact which is sufficient to a transfer to transfer HCV present in the sample into the body of the subject and thereby infecting the subject with HCV.

The suitable amount of water-soluble Agent to reduce the likelihood that a subject infected with HCV can, according to the skilled person be determined by known methods. In one embodiment the appropriate amount of water-soluble agent is an amount between about 1 pM and about 10 mM. In the preferred embodiment the appropriate amount of water-soluble agent is an amount between about 1 pμ and about 10 μM.

In an embodiment If the agent is an antibody. In a further embodiment if the agent is a DC-SIGN and / or DC-SIGNR based molecule.

The The present invention further discloses a method of treatment a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HIV-1, essential is reduced, comprising the steps of (a) contacting the Sample with a suitable amount of a water-soluble agent, which is able to complex with in a specific way one on an HCV envelope glycoprotein existing domain to form, so that a complex between the agent and HCV, provided present in the sample is formed; and (b) removing any thus formed complex of the resulting sample, so that This reduces the probability that a subject infected with HCV as a result of contact with the sample.

The Removal of the complex from the resulting sample can be done according to the person skilled in the art well-known methods take place. Such methods include, for. B. affinity one.

The inventive method may further include the step of removing uncomplexed Include agent from the sample if such removal is desirable is (for example if the agent is unwanted Effects in a subject to which it is administered becomes).

The The present invention further discloses a method of treatment a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising the steps of (a) contacting the sample with a suitable amount of a water-soluble agent which (i) capable of doing so in a specific way a complex with one on an HCV envelope glycoprotein existing domain and (ii) comprises a residue capable of doing so is, specifically, a complex with a known ligand the remainder being the removal of the agent from a sample allowed by the sample with an immobilized form the known ligand is contacted, leaving a complex formed between the agent and HCV, if present in the sample becomes; and (b) removing any complex so formed from the resulting sample by contacting the resulting Sample with an immobilized form of the known ligand, so that thereby reduces the probability that a subject infected by HCV as a result of contact with the sample.

method immobilization of a ligand is well known to those skilled in the art. As used herein, a ligand in its "immobilized form" is capable of a complex with that specific by the ligand in its free form to form recognized remainder.

The The present invention further discloses a method of treatment a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising, comprising the steps of (a) contacting the sample suitable conditions with an article of manufacture comprising a solid support, on which an agent is surgically attached, which in the Location is, in a specific way, a complex with one on an HCV envelope glycoprotein existing domain to build; and (b) contacting the sample with a suitable one Amount of a water-soluble Agent that is capable of doing so in a specific way a complex with an HCV envelope glycoprotein present domain to form, so that a complex between the agent and HCV, provided present in the sample, is formed, on the condition that Step (a) can be done either before or after step (b).

The The present invention further discloses a method of treatment a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising, comprising the steps of (a) contacting the sample suitable conditions with an article of manufacture comprising a solid support, on which an agent is surgically attached, which in the Location is, in a specific way, a complex with one on an HCV envelope glycoprotein existing domain to build; and (b) (i) contacting the sample with a suitable one Amount of a water-soluble Agens, which is capable of, in a specific way a Complex with an HCV envelope glycoprotein present domain so as to form a complex between the agent and HIV-1, provided present in the sample, and (ii) removing any thus formed complex from the resulting sample, under the condition that step (a) either before or after step (b) can be done.

The present invention further discloses a method of treating a body fluid sample such that the likelihood of a subject becoming infected with HCV as a result of contact with the sample is substantially reduced, comprising the steps of (a) contacting the sample under suitable conditions with an article of manufacture comprising a solid support to which an agent operatively attached which is capable of specifically forming a complex with a domain present on an HCV envelope glycoprotein; and (b) (I) contacting the sample with a suitable amount of a water-soluble agent which (1) is capable of specifically forming a complex with a domain present on an HIV-1 envelope glycoprotein and ( 2) comprises a moiety which is capable of specifically forming a complex with a known ligand, thereby forming a complex between the agent and HCV, if present in the sample, and (II) removing any such complex formed from the resulting sample by contacting the resulting sample with an immobilized form of the known ligand, under the condition that step (a) can be performed either before or after step (b).

The Methods of the present invention may further include the step removal of target cells from the body fluid sample. In one embodiment the target cells are leukocytes. Procedure for Removal of leukocytes from a body fluid sample will be apparent to those skilled in the art well known and close z. B. leucofiltration.

As used herein, a body fluid is any one Liquid, which in the body a subject exists and is able to HCV in one to contain HCV infected subject. Body fluids include, but are not limited whole blood or derivatives thereof (eg, red blood cell preparations and platelets), Saliva, spinal fluid, Tears, Vaginal secretions, urine, alveolar fluid, synovial fluid, Sperm, pleural fluid and bone marrow. In the preferred embodiment, it is in the body fluid to a liquid, which is to be administered to a subject. Also is in the preferred embodiment the body fluid sample selected from the group consisting of whole blood, a preparation from red blood cells, a preparation from platelets as well as sperm.

The Body fluid samples as whole blood can further include exogenous substances which may be of clinical or Storage purposes were added. Such exogenous substances include, for. B. Anticoagulants (eg citrate) and preservatives (eg. Dextrose).

In an embodiment become the steps of contacting the methods of the present invention at about 4 ° C carried out. In a further embodiment The steps of contacting the methods of the present invention will be described Invention at about 20 ° C. carried out. In yet another embodiment The steps of contacting the methods of the present invention will be described Invention carried out at about 37 ° C.

The Invention also discloses a kit for treating a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising the previously described article of manufacture.

The Invention further discloses a kit for treating a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising, in separate sections: (a) an article of manufacture comprising a solid support, on which an agent is surgically attached, which in the Location is, in a specific way, a complex with one on an HCV envelope glycoprotein existing domain to build; and (b) a water-soluble Agent that is capable of doing so in a specific way a complex with an HCV envelope glycoprotein present domain to build.

The Invention further discloses a kit for treating a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, significantly reduced comprising, in separate sections: (a) an article of manufacture comprising a solid support, on which an agent is surgically attached, which in the Location is, in a specific way, a complex with one on an HCV envelope glycoprotein existing domain to build; and (b) a water-soluble Agent which (1) is capable of, in a specific way and Make a complex with an HCV envelope glycoprotein present domain and (2) comprises a remainder which is capable of forming is, in a specific way, a complex with a known one Forming ligands, said residue removing the agent from a sample over contacting with an immobilized form of the known Ligands allowed; and (c) comprising an article of manufacture a solid support, to which the known ligand is operatively attached, which in addition is able to complex with in a specific way the rest of the water-soluble Agent of step (b) to form.

The Invention discloses a kit for treating a body fluid sample, allowing the probability of a subject as a result the contact with the sample is infected with HCV, is significantly reduced, comprising, in separate sections: (a) a water-soluble Agent which (i) is capable of, in a specific way and Make a complex with an HCV envelope glycoprotein present Domain too and (ii) comprises a moiety which is capable of in a specific way a complex with a known one Forming ligands, said residue removing the agent from a sample over contacting with an immobilized form of the known Ligands allowed; and (b) comprising an article of manufacture a solid support, to which the known ligand is operatively attached, which in addition is able to complex with in a specific way the rest of said water-soluble To form agent.

The The present invention also discloses a kit for reduction that in a body fluid sample present amount of HCV or HCV envelope glycoprotein, comprising the previously described article of manufacture. In one embodiment it is the body fluid for blood.

The Kits of the present invention may further be suitable Buffer include.

Around the understanding The following examples are intended to facilitate certain often occurring methods and / or terms best in Sambrook et al. described.

The Methods for trapping HCV virions described herein may be used be used for any purpose known in the art. In one embodiment the procedure is applied in such a way that the infectivity of a Sample of a subject is reduced. In one embodiment the method for concentrating the HCV virions is used, so the odds for to increase the detection of HCV, so as in a PCR assay for HCV nucleic acid, as well as HCV RNA.

Obtaining a sample of HCV envelope glycoprotein ⁺ cells may be carried out according to methods well known to those skilled in the art. HCV envelope glycoprotein ⁺ cells can be obtained from blood or any other body fluid known to contain HCV envelope glycoprotein ⁺ cells in HCV-infected subjects.

The present invention discloses a compound or agent which is capable of binding a DC-SIGN protein to an HCV envelope glycoprotein to inhibit HCV infection of a cell is inhibited. The present invention provides a compound or agent, which is capable of binding a DC-SIGNR protein to an HCV envelope glycoprotein to inhibit HCV infection of a cell is inhibited.

The The present invention discloses an antibody or a part thereof which is capable of binding a DC-SIGN protein to an HCV envelope glycoprotein to inhibit, said antibody binds to an epitope, which is within a region of the DC-SIGN protein, wherein said region of the DC-SIGN protein to an HCV envelope glycoprotein binds. The present invention provides an antibody or a part of it ready, which is capable of binding of a DC-SIGNR protein to an HCV envelope glycoprotein, wherein said antibody binds to an epitope which is within a region of the DC-SIGNR protein wherein said region of the DC-SIGNR protein is linked to an HCV envelope glycoprotein binds.

The The present invention discloses an antibody or a part thereof which is capable of binding a DC-SIGN protein to an HCV envelope glycoprotein to inhibit, said antibody binds to an epitope, which is within a region of the HCV envelope glycoprotein, wherein the said region of the HCV envelope glycoprotein binds to a DC-SIGN protein. The present invention provides an antibody or part of it ready, which is capable of, the Inhibit binding of a DC-SIGN protein to an HCV envelope glycoprotein, wherein said antibody binds to an epitope which is within a region of the HCV envelope glycoprotein wherein said region of the HCV envelope glycoprotein binds to a DC-SIGNR protein.

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or parts thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies described herein or parts thereof, han the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises an F (ab ') ₂ portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises an Fd portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises an Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

In an embodiment the antibody described herein or parts thereof, the antibody binds to an epitope, which within a region of an E1-HCV envelope glycoprotein lies. In one embodiment of the Antibodies described herein or parts thereof, the antibody binds to an epitope, which is within a region of an E2-HCV envelope glycoprotein.

The Invention includes antibodies or fragments of antibodies, which the ability possess the interaction between HCV and DC-SIGN and / or the Block interaction between HCV and DC-SIGNR. The antibodies can be used for HCV, DC-SIGN or DC-SIGNR. According to another aspect of the invention becomes an antibody with the aforementioned specificity for use in the treatment of any HCV infection and in the manufacture of a medicament for the treatment of HCV infection provided. For the antibody it is preferably a monoclonal antibody. One such antibodies can be used to temporarily block the DC-SIGNR receptor and avoid infection by HCV, e.g. B. immediately after an accidental infection by HCV-infected blood.

As used herein, "antibody" includes both naturally occurring ones as well as not natural occurring antibodies one. In particular, "antibody" includes polyclonal and monoclonal antibodies and monovalent and divalent fragments thereof. Furthermore includes "antibodies" chimeric antibodies, completely synthetic Antibody, Single-chain antibody as well as fragments thereof. The antibody may be a human or a non-human antibody. A nonhuman antibody can be humanized by recombinant methods to obtain its immunogenicity to reduce in humans. antibody be according to the conventional Methodology made. Monoclonal antibodies can be generated using the method Kohler and Milstein (Nature, 256: 495, 1975). For the production of monoclonal antibodies useful according to the invention a mouse or other suitable host animal at appropriate intervals (eg every two weeks, weekly, twice a month or monthly) with antigenic forms of HCV, HCV envelope glycoproteins, DC-SIGN or DC-SIGNR immunized. The animal may be in the last Week before sacrificing a final "boost" of antigen administered it is desirable while immunization using an immunological adjuvant. suitable immunological adjuvants include: complete Freund's Adjuvant, Freund's incomplete adjuvant, alum, RIBI adjuvant, Hunter's TiterMax, Saponin adjuvants such as QS21 or Quil A or CpG containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in the art. The animals can be transmitted via subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes are immunized. A certain Animal can be immunized with multiple forms of the antigen by multiple routes become.

In an embodiment HCV is used from the plasma of HCV-infected individuals the process of sucrose gradient centrifugation. Alternatively, recombinant HCV E1 and / or E2 envelope glycoproteins consisting of a variety of sources, such as Austral Biologicals (San Ramon, CA., Cat. # HCA-090-2), Immunodiagnostics (Woburn, MA, USA, Cat. # 4001) and Accurate Chemical (Westbury, MA, USA, cat # YVS8921). The recombinant HCV envelope glycoproteins can by surface expression provided on recombinant cell lines. DC-SIGN can provided in the form of human dendritic cells, whereas DC-SIGNR is provided in the form of liver sinusoidal cells can be. Recombinant forms of DC-SIGN and DC-SIGNR can be found under Use of the previously described methods (Pohlmann, Soilleux et al., 2001, ID: 1081). Alternatively, it can the antigen is provided in the form of synthetic peptides, which relevant antigenic regions correspond.

According to the immunization regime, lymphocytes from the spleen, from lymph nodes or out isolated from another organ of the animal and fused with an appropriate myeloma cell line using an agent such as polyethylene glycol to form a hybridoma. Following fusion, the cells are placed in media using standard techniques that allow growth of the hybridomas, but not the fusion partners, as described (Goding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology. Biochemistry and Immunology, 3rd Edition, Academic Press, New York, 1996).

in the Following the cultivation of the hybridomas, the cell supernatants are the presence of antibodies with the desired specificity, d. H. which selectively bind the antigen are analyzed. suitable Close analysis techniques a: ELISA, flow cytometry, immunoprecipitation and Western blotting. Further Screening techniques are well known in the art. preferred Techniques are those involving the binding of antibodies confirm conformationally intact, natively folded antigen, so such as non-denaturing ELISA, flow cytometry and immunoprecipitation.

Importantly, as is well known in the art, only a small portion of the antibody molecule, paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, WR (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons , Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Edition, Blackwell Scientific Publications, Oxford). The pFc 'and the Fc region are z. B. effectors of the complement cascade, but are not involved in the antigen binding. An antibody from which the pFc 'region has been enzymatically cleaved or which has been produced without the pFc' region, referred to as the F (ab ') ₂ fragment, retains both antigenic binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved or which has been produced without the Fc region, referred to as a Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. In addition, Fab fragments consist of a covalently linked antibody light chain and a portion of the antibody heavy chain designated Fd. The Fd fragments are the major determinants of antibody specificity (a single Fd fragment can be up to ten different light chains are associated with it without altering the specificity of the antibody) and they retain their epitope binding capabilities in isolation.

Within of the antigen-binding portion of an antibody exist as in the prior art well known in the art, Complementarity Determining Regions (CDRs), which interact directly with the epitope of the antigen, as well as framework regions (FRs) which are the tertiary Maintain structure of the paratope (see generally Clark, 1986; Roitt, 1991). In the Fd fragment of both the heavy chain and There are four framework regions in the light chain of IgG immunoglobulin (FR1 to FR4), each of which is defined by three complementarity determining Regions (CDR1 to CDR3) are separated. The CDRs, and especially the CDR3 regions, and more specifically the CDR3 heavy chain, are for the most part responsible for the specificity of the antibody.

It has now become well established in the art that the non-CDR regions of a mammalian antibody by similar Replaces regions of conspecific or heterospecific antibodies can be and the epitope specificity of the original one antibody maintained. This is most evident in the development and the use of "humanized" antibodies, in which non-human CDRs are covalently bound to human FR and / or Fc / pFc 'regions to produce a functional antibody.

In certain embodiments, the present invention provides compositions and methods that include humanized forms of antibodies. As used herein, "humanized" refers to those antibodies in which some, most or all, of the amino acids outside the CDR regions are replaced by corresponding amino acids derived from human immunoglobulin molecules U.S. Patents 4,816,567 . 5,225,539 . 5,585,089 . 5,693,761 . 5,693,762 and 5,859,205 but are not limited to these.

In one embodiment of the humanized forms of antibodies, some, most or all, of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules, but some, most or all, of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not override the ability of the antibody to bind a particular antigen. Suitable human immunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains similar specificity as the original antibodies. However, using certain methods of humanization, using the "directed evolution" methods, as described by Wu et al., J. Mol. Biol. 294: 151, 1999, the binding affinity and / or specificity of the antibody can be increased.

Fully human monoclonal antibodies can also be generated by immunizing mice that are transgenic to large parts of the human immunoglobulin heavy and light chain loci. See, for example, B. the U.S. Patents 5,591,669 . 5,598,369 . 5,545,806 . 5,545,807 . 6.150584 as well as the references cited therein. These animals have been genetically modified to have a functional deletion in the production of endogenous (eg murine) antibodies. Furthermore, the animals were modified to contain all or part of the human germ-line immunoglobulin gene locus such that immunization of these animals would result in the production of fully human antibodies to the relevant antigen. Following immunization of these mice (eg, XenoMouse (Abgenix), HuMAb mice (Medarex / Genfharm)), monoclonal antibodies can be generated according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and will therefore not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

There are also in vitro methods for producing human antibodies. These include the phage display technology ( U.S. Patents 5,565,332 and 5,573,905 ) and the in vitro stimulation of human B cells ( U.S. Patents 5,229,275 and 5,567,610 ) one.

As will be apparent to those skilled in the art, the present invention also provides: F (ab ') ₂ , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F (ab ') ₂ fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.

The different antibody molecules and fragments can any of the commonly known immunoglobulin classes deduce, including, but not limited on: IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are The person skilled in the art also well known and include, but are not limited on: IgG1, IgG2, IgG3 and IgG4.

monoclonal antibody can by mammalian cell cultures be generated in hybridoma or recombinant cell lines, such as Ovarian cells of Chinese hamsters or murine myeloma cell lines. Such methods are well known to those skilled in the art. It can too Bacterial, yeast and insect cell lines are used monoclonal antibodies or to produce fragments thereof. There are also procedures for Generation of monoclonal antibodies in transgenic animals or plants (Pollock et al., J. Immunol. Methods, 231: 147, 1999; Russell, Curr. Top. Microbiol. Immunol. 240: 119, 1999).

In one embodiment of the agents described herein, the agent is an antibody or part of an antibody. As used herein, "antibody" refers to an immunoglobulin molecule comprising two heavy chains and two light chains and recognizing an antigen The immunoglobulin molecule can be derived from any of the commonly known classes, including, but not limited to, IgA, secretory IgA, IgG, and IgM IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4, including, for example, both naturally occurring and non-naturally occurring antibodies "Antibodies" include polyclonal and monoclonal antibodies as well as monovalent and divalent fragments thereof. In addition, "antibody" includes chimeric antibodies, fully synthetic antibodies, single chain antibodies, and fragments thereof Optionally, an antibody can be labeled with a detectable marker Detectable labels include, for example, radioactive or fluorescent labels A non-human antibody can be humanized by recombinant methods to reduce its immunogenicity in humans Methods for humanizing antibodies are known to those of skill in the art As used herein, "monoclonal antibody", also referred to as mAb called, antibody molecules whose primary sequences are substantially identical and which have the same antigen specificity exhibit. Monoclonal antibodies can be generated by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art. The term "antibody" includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies, In particular, the term "antibody" includes polyclonal and monoclonal antibodies as well as antigen-binding fragments thereof. Further, the term "antibody" includes chimeric antibodies, fully synthetic antibodies, and antigen-binding fragments thereof Accordingly, in one embodiment, the antibody is a monoclonal antibody In one embodiment, the antibody is a polyclonal antibody In one embodiment, the antibody is a humanized antibody In one embodiment, the antibody is a chimeric antibody Such chimeric antibodies may include one part of an antibody from one source and part of an antibody from another source ,

In one embodiment, the part of the antibody comprises a light chain of the antibody. As used herein, "light chain" refers to the smaller polypeptide of an antibody molecule composed of a variable domain (VL) and a constant domain (CL) or fragments thereof In one embodiment, the portion of the antibody comprises a heavy chain of the antibody. As used herein, "heavy chain" refers to the larger polypeptide of an antibody molecule composed of a variable domain (VH) and three or four constant domains (CH1, CH2, CH3 and CH4) or fragments thereof. In one embodiment, the portion of the antibody comprises a Fab portion of the antibody. As used herein, "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of a light chain and a portion of a heavy chain It can be obtained by short papain digestion or by recombinant methods F (ab ') ₂ portion of the antibody As used herein, "F (ab') 2 fragment" refers to a bivalent antigen binding fragment of an immunoglobulin consisting of both light chains and a portion of both heavy chains. It can be obtained by short pepsin digestion or by recombinant methods. In one embodiment, the portion of the antibody comprises an Fd portion of the antibody. In one embodiment, the portion of the antibody comprises an Fv portion of the antibody. In one embodiment, the portion of the antibody comprises a variable domain of the antibody. In one embodiment, the portion of the antibody comprises a constant domain of the antibody. In one embodiment, the portion of the antibody comprises one or more CDR domains of the antibody. As used herein, "CDR" or "complementarity determining region" refers to a highly variable sequence of amino acids in the variable domain of an antibody.

The The present invention discloses humanized forms of those described herein Antibody. As used herein, "humanized" refers to antibodies, in which some, most or all of the amino acids outside the CDR regions through replaced corresponding amino acids derived from human immunoglobulin molecules were. In one embodiment The humanized forms of antibodies have been some, most or all of the amino acids outside CDR regions derived from human immunoglobulin molecules amino acids but some, most or all of the amino acids remained within one or more CDR regions unchanged. Small additions, deletions, Insertions, substitutions or modifications of amino acids permissible as long as she does not have the ability of the antibody override set to bind a particular antigen. Suitable human immunoglobulin molecules would be IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules lock in. A "humanized" antibody would be a similar one specificity like the original one antibody maintained.

It is known to those skilled in the art how to generate the humanized antibodies of the present invention. Various publications, some of which are hereby incorporated by reference in the present application, also describe the production of humanized antibodies. To include z. B. the in U.S. Patent No. 4,816,567 describe the production of chimeric antibodies with a variable region of one antibody and a constant region of another antibody.

U.S. Patent No. 5,225,539 describes another approach to the generation of a humanized antibody. This patent describes the use of recombinant DNA technology to produce a humanized antibody in which the CDRs of an immunoglobulin variable region are replaced by CDRs of an immunoglobulin of a different specificity such that the humanized antibody would recognize the desired target, but itself would not be detected in a significant way by the subject's immune system. More specifically, site-directed mutagenesis is used to transfer the CDRs to the framework.

Further approaches to the humanization of an antibody are described in the U.S. Patents Nos. 5,585,089 and 5,693,761 as in WO 90/07861 which describe processes for the preparation of humanized immunoglobulins. These have one or more CDRs and possible additional amino acids from a donor immunoglobulin as well as a framework region from a human acceptor immunoglobulin. The cited patents describe a method for increasing the affinity of an antibody for the desired antigen. Some amino acids in the backbone are chosen to be more identical to the amino acids at the given positions in the donor than in the acceptor. More specifically, these patents describe the generation of a humanized antibody that binds to a receptor by combining the CDRs of a murine monoclonal antibody with human immunoglobulin framework and constant regions. Human framework regions can be selected to maximize homology to the murine sequence. A computer model can be used to identify the amino acids in the framework region that are likely to interact with the CDRs on the specific antigen, and then at these positions murine amino acids can be used to generate the humanized antibody.

The aforementioned patents 5,585,089 and 5,693,761 and WO 90/07861 also suggest four possible criteria which may find application in the production of humanized antibodies. The first suggestion was to use for the acceptor a scaffold of a particular human immunoglobulin exhibiting unusually high homology to the donor immunoglobulin to be humanized, or a consensus scaffold of many human antibodies. The second suggestion was that if an amino acid in the human immunoglobulin framework is unusual and the donor amino acid at that position is typical of human sequences, the donor amino acid can be selected rather than the acceptor. The third suggestion was that at the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid can be selected rather than the acceptor amino acid. The fourth proposal was to use the donor amino acid residue at the framework positions at which it is predicted that the amino acid in a three-dimensional model of the antibody has a side chain atom within 3 Å of the CDRs and is able to interact with the CDRs , The foregoing methods are merely illustrative of some of the methods of producing humanized antibodies that one skilled in the art might use.

The The present invention discloses isolated nucleic acids which encode the agents and / or compounds described herein. In one embodiment encodes the nucleic acid the antibodies described herein or their humanized versions. In the nucleic acid can it may be RNA, DNA or cDNA. In one embodiment encodes the nucleic acid the light chain. In one embodiment, encoded the nucleic acid the heavy chain. In one embodiment encodes the nucleic acid both the heavy and the light chain. In one embodiment One or more nucleic acids encode the Fab portion. In a embodiment One or more nucleic acids encode the CDR sections. In a embodiment encodes the nucleic acid the variable domain.

The The present invention discloses the nucleic acids described herein, wherein the nucleic acids through the insertion, deletion and / or substitution of one or more Nucleotides changed can be what to a change the nucleic acid sequence to lead could. In one embodiment to lead the nucleotide changes not to a mutation at the amino acid level. In one embodiment can change the nucleotide to an amino acid change to lead. With such an amino acid change it could be one that does not affect the function of the protein.

The The present invention discloses a vector which includes one herein described nucleic acid includes. In one embodiment If the vector is a plasmid. The present invention provides a host-vector system which is as described herein Vector and a suitable host cell. The present invention provides a method for producing a polypeptide comprising breeding of the host-vector system described herein under conditions, which for the production of the polypeptide and to obtain the thus prepared Polypeptids are suitable.

In an embodiment The agents described herein are the agent to a polypeptide. In one embodiment The agents described herein are the agent to an oligopeptide. As used herein, "polypeptide" refers to two or more linked by a peptide bond Amino acids.

The present invention discloses a polypeptide which is capable of inhibiting the binding of a DC-SIGN protein to an HCV envelope glycoprotein, said polypeptide having consecutive amino acids having a sequence corresponding to the sequence of at least a portion of an extracellular domain of a DC-SIGN protein, which part binds to an HCV envelope glycoprotein.

In an embodiment the polypeptide corresponds to an extracellular domain of DC-SIGN. In one embodiment of the polypeptide, the extracellular domain comprises consecutive amino acids a sequence which starts with the lysine at position 62 and with the carboxyterminal amino acid as shown in SEQ ID NO: 1 ends.

In an embodiment of the polypeptide, the extracellular domain is a lectin binding domain Lectin domain Type C or part of it.

In an embodiment of the polypeptide comprises the C-type lectin domain, consecutive amino acids a sequence starting with the leucine at position 229 and with the carboxyterminal amino acid as shown in SEQ ID NO: 1 ends.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGNR protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence which corresponds to the sequence of at least one part an extracellular domain a DC-SIGNR protein corresponds, wherein the part binds to an HCV envelope glycoprotein. In a Embodiment of the Polypeptides include the extracellular domain with consecutive amino acids a sequence which starts with the lysine at position 74 and with the carboxyterminal amino acid as shown in SEQ ID NO: 2 ends.

In an embodiment of the polypeptide comprises the C-type lectin domain, consecutive amino acids a sequence which starts with the leucine at position 241 and with the carboxyterminal amino acid as shown in SEQ ID NO: 2 ends.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGN protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence comprising the sequence of at least one part an extracellular domain an HCV envelope glycoprotein corresponds, wherein the part binds to a DC-SIGN protein.

In an embodiment For example, the polypeptide comprises consecutive amino acids having a sequence which the sequence of at least part of the extracellular domain of a E1 HCV envelope glycoprotein corresponds, wherein the part binds to a DC-SIGN protein. In a embodiment For example, the polypeptide comprises consecutive amino acids having the amino acid sequence as shown in SEQ ID NR: 3 sequence shown from position 192 to position 346 or a part of it.

In an embodiment For example, the polypeptide comprises consecutive amino acids having a sequence which the sequence of at least part of the extracellular domain of a E2 HCV envelope glycoprotein corresponds, wherein the part binds to a DC-SIGN protein. In a embodiment For example, the polypeptide comprises consecutive amino acids having the amino acid sequence as shown in SEQ ID NR: 3 sequence shown from position 383 to position 717 or a part of it.

The The present invention discloses a polypeptide useful in capable of binding a DC-SIGNR protein to an HCV envelope glycoprotein with the polypeptide having consecutive amino acids a sequence comprising the sequence of at least one part an extracellular domain an HCV envelope glycoprotein corresponds, wherein the part binds to a DC-SIGNR protein.

In an embodiment For example, the polypeptide comprises consecutive amino acids having a sequence which the sequence of at least part of the extracellular domain of a E1 HCV envelope glycoprotein corresponds, wherein the part binds to a DC-SIGNR protein. In a embodiment For example, the polypeptide comprises consecutive amino acids having the amino acid sequence as shown in SEQ ID NR: 3 sequence shown from position 192 to position 346 or a part of it.

In an embodiment For example, the polypeptide comprises consecutive amino acids having a sequence which the sequence of at least part of the extracellular domain of a E2 HCV envelope glycoprotein corresponds, wherein the part binds to a DC-SIGNR protein. In a embodiment For example, the polypeptide comprises consecutive amino acids having the amino acid sequence as shown in SEQ ID NR: 3 sequence shown from position 383 to position 717 or a part of it.

The Compounds and / or agents described herein can be used by Any means known in the art can be prepared. So can z. As a protein by recombinant expression from a nucleic acid, so as a plasmid or a nucleic acid encoding the Vector, wherein the plasmid or the vector is in a suitable host cell, i. H. a host-vector system for manufacturing of the relevant polypeptide. A suitable vector can be produced which are suitable regulatory sequences as well as enhancers and promoters. The host cell may be a any cell type, including mammalian, bacterial and yeast cells, but not limited to these. Suitable bacterial cells include E. coli cells. suitable mammalian cells shut down but are not limited human embryonic kidney (HEK) 293T cells, HeLa cells, NIH 3T3 cells, Chinese hamster ovary cells (CHO cells) and Cos cells.

Becomes The protein produced recombinantly, it may be from a plasmid containing a synthetic nucleic acid insert are expressed. Such an insertion site in the plasmid may be the linkage of the protein with one day, such as a poly histidine tag. Such Day allows the later one Protein purification.

A Nucleic acid, which is the polypeptide, protein or functional equivalent thereof encoded under the control of an inducible promoter be cloned, allowing regulation of protein expression becomes. Suitable inducible systems are known to the person skilled in the art.

vectors for expression of the protein or the functional equivalent described herein of it can selected from commercial sources be or for a particular expression system can be constructed. Such vectors can contain suitable regulatory sequences, such as promoter sequences, Terminator sequences, polyadenylation sequences, enhancer sequences and marker genes. Vectors may be plasmids or you can based on viruses. The skilled person can do this Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989). Lots known techniques and protocols for the manipulation of nucleic acids and the analysis of proteins are described in detail in "Short protocols in molecular biology ", second edition, Ausubel et al. (John Wiley & Sons, 1992).

method for isolation and purification of recombinant proteins known to those skilled in the art and are described in various sources, as in Sambrook et al. (1989). In bacteria like E. coli can the recombinant protein within the bacterial cell inclusion bodies form, whereby its production is facilitated. Will it be in inclusion bodies produced, so can the carrier protein a refolding in a natural Need conformation.

Additionally, for adapting the properties of the protein or the functional equivalent thereof, recognizes the expert that at the nucleic acid level changes can be made of known protein sequences, so as by addition, substitution, deletion or insertion of a or more nucleotides. In site-directed mutagenesis it is the preferred method for producing mutated Proteins. Those skilled in the art will find many site-oriented techniques Mutagenesis known, including but not limited to oligonucleotide-directed mutagenesis using PCR, as described in Sambrook or using commercially available Kits.

It can selected suitable vectors or constructed, containing appropriate regulatory sequences, inclusively Promoter sequences, polyadenylation sequences, enhancer sequences, marker genes and more sequences, depending on their suitability. The vectors include, but are not limited on: plasmids, such as viral plasmids, z. Phage plasmids, or Phagemid and as described in Sambrook. Techniques and protocols for the manipulation of nucleic acids, as in the production of nucleic acid constructs, in mutagenesis, when sequencing, at the introduction of nucleic acids in cells and gene expression as well as in the analysis of proteins are in "Short protocols in molecular biology ", second Edition, Ausubel et al. (John Wiley & Sons, 1992).

The The present invention also discloses soluble forms of those described herein Polypeptides. Accordingly, z. B. a transmembrane domain for a on a cell surface expressed polypeptide, so that the polypeptide soluble would become.

The present invention discloses a non-peptidyl agent capable of inhibiting the binding of a DC-SIGN protein to an HCV envelope glycoprotein, said non-peptidyl agent binding to an epitope which is within a region of the DC. SIGN protein, said region of the DC-SIGN protein binding to an HCV envelope glycoprotein. The present invention provides a non-pepti A dyl agent capable of inhibiting the binding of a DC-SIGNR protein to an HCV envelope glycoprotein, wherein said non-peptidyl agent binds to an epitope located within a region of the DC-SIGNR protein wherein said region of the DC-SIGNR protein binds to an HCV envelope glycoprotein.

The The present invention discloses a non-peptidyl agent which capable of binding a DC-SIGN protein to a HCV envelope glycoprotein to inhibit, wherein the non-peptidyl agent at least one Section of an extracellular domain an HCV envelope glycoprotein binds, said portion binding to a DC-SIGN protein. The present invention provides a non-peptidyl agent, which is capable of binding a DC-SIGNR protein an HCV envelope glycoprotein to inhibit, wherein the non-peptidyl agent at least to a section an extracellular domain an HCV envelope glycoprotein binds, said portion binds to a DC-SIGNR protein.

In an embodiment The nonpeptidyl agent described herein binds the nonpeptidyl agent to at least a portion of an extracellular domain of an E1-HCV envelope glycoprotein. In one embodiment The nonpeptidyl agent described herein binds the nonpeptidyl agent to at least a portion of an extracellular domain of an E2-HCV envelope glycoprotein.

In an embodiment The nonpeptidyl agent described herein is included the nonpeptidyl agent is a carbohydrate. At the carbohydrate it may be a carbohydrate known to those skilled in the art, including, but not limited on, mannose, mannan and methyl α-D-mannopyranoside.

As As used herein, "non-peptidyl agent" refers to an agent which not in its entirety from a linear sequence of Linked peptide bonds amino acids consists. A nonpeptidyl molecule however, may contain one or more peptide bonds. In a embodiment when the non-peptidyl agent is a compound with a molecular weight of less than 500 daltons. As used herein denotes a "small Molecule "or a molecule with low Molecular weight one molecule with a molecular weight of less than 500 daltons.

The The present invention discloses a composition comprising a carrier and a compound which inhibits the binding of HCV to DC-SIGN and / or DC-SIGNR on the surface a cell is inhibited. In one embodiment, the composition comprises an amount of the compound which is sufficiently effective, the binding from HCV to DC-SIGN and / or DC-SIGNR to the surface of a Cell to inhibit.

The The present invention discloses a composition comprising a Antibodies described herein or part thereof and a carrier. The present invention provides a composition comprising one described herein Polypeptide and a carrier. The present invention provides a composition comprising a nonpeptidyl agent and a carrier described herein. The carrier shut down but are not limited on: an aerosol, intravenous, oral and topical carriers. Accordingly, the present invention provides the aforementioned Composition ready for which Aerosol, intravenous, oral or topical applications or others known to those skilled in the art Applications has been adapted.

The present invention discloses the agents described herein Compounds and / or compositions and a carrier. at such a carrier it may be a pharmaceutically acceptable carrier. pharmaceutical compatible carrier are well known to those skilled in the art. Such pharmaceutically acceptable carrier can lock in, but are not limited in: watery or non-aqueous Solutions, Suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, Polyethylene glycol, vegetable oils like olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include: Water, alcoholic / watery Solutions, Emulsions or suspensions, saline and buffered media. parenteral Close the vehicle a: sodium chloride solution, Ringer's Dextrose, Dextrose and Sodium Chloride, lactated Ringer's or fixed oils. intravenous Vehicles include: liquid and nourishing Replenisher, electrolyte replenisher, as well as Ringer's Dextrose based, and the like. It can also contain preservatives and other additives, such as e.g. B. antimicrobial Substances, antioxidants, chelators, inert gases and the like.

As used herein, "composition" refers to a mixture The compositions include those which are oral, rectal, intravaginal, topical, nasal, ophthalmic or parent are suitable for administration to a subject but are not limited thereto. As used herein, "parenteral" includes, but is not limited to, subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques As used herein, "administration" may be done or performed using any method known to those skilled in the art. The methods of administration to a subject include, but are not limited to, oral, rectal, intravaginal, topical, nasal, ophthalmic, parenteral, subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques.

The The present invention discloses DC-SIGN and DC-SIGNR proteins or functional equivalents thereof for use in the therapy or diagnosis of HCV. The invention provides a connection for use in therapy or diagnosis of HCV which specifically binds to DC-SIGN and / or DC-SIGNR proteins.

As used herein is a functional equivalent from DC-SIGN or DC-SIGNR to a connection that is capable of HCV to bind, reducing their interaction with DC-SIGN and / or DC-SIGNR is prevented. Preferably, the functional equivalent is to a peptide or protein. The term "functional equivalent" includes fragments, Mutants and muteins of DC-SIGN and DC-SIGNR. Functional equivalents shut down molecules which bind HCV, preferably the HCV envelope glycoproteins, and all or a part of the extracellular domains from DC-SIGN or DC-SIGNR.

The functional equivalents shut down soluble Forms of DC-SIGN or DC-SIGNR proteins one. A suitable soluble Form of these proteins or functional equivalents thereof could e.g. B. a shortened form of the protein from which the transmembrane domain passes chemical, proteolytic or recombinant method was removed. The transmembrane domain DC-SIGN starts at about glycine 49 and ends at about serine 61, whereas the transmembrane domain of DC-SIGNR is approximately glycine 49 begins and ends at about Serin 73.

In an embodiment includes the functional equivalent all or part of the extracellular domain of DC-SIGN or DC-SIGNR. The extracellular Region of DC-SIGN starts at about lysine 62 and closes the carboxy terminal amino acids whereas the extracellular Region of DC-SIGNR starting at lysine 74 and enclosing the carboxyterminal amino acids.

The functional equivalent is preferably at least 80% homologous to the corresponding protein. In a preferred embodiment is the functional equivalent at least 90% homologous, according to the rating by any common Analysis algorithm, such as. For example, the Pileup Sequence Analysis software (Program Manual for the Wisconsin Package, 1996). The numbering of the amino acids takes place as in the GenBank protein with accession number AAK20997 for DC-SIGN and AAG13848 for DC-SIGNR provided.

Of the Term "a functionally equivalent Fragment "can, as used herein, also any fragment or a any composition of fragments of DC-SIGN and / or DC-SIGNR which is linked to HCV, preferably to the HCV envelope glycoproteins, binds. The C-type lectin binding domain of DC-SIGN starts at about leucine 229 and includes the carboxyterminal amino acids, whereas the C-type lectin binding domain of DC-SIGNR is approximately leucine 241 begins and includes the carboxy-terminal amino acids. The full Protein, the extracellular domain or the C-type lectin domain can shortened one or both ends be or can internally sections are removed, provided that the protein retains the defined function.

Proteinaceous, functionally equivalent Fragments or analogs can belong to the same protein family as those identified herein human DC-SIGN and DC-SIGNR proteins. With "protein family" is a group of proteins that have a common function and show a common sequence homology. Homologous proteins can take off derived from non-human species. Preferably, the homology is between functionally equivalent Protein sequences at least 25% over the entirety of the amino acid sequence of the complete Protein or the whole EC2 fragments (amino acids 11.3-201). More preferably the homology at least 50%, even more preferably 75% over the Entirety of the amino acid sequence of the protein or protein fragment. More preferred is homology greater than 80% over the entire sequence. More preferably, the homology is greater than 90% over the entire sequence. More preferably, the homology is greater than 95% over the entire sequence.

Of the Term "a functionally equivalent Analogue "is used to describe the compounds that have an analogous function to an activity have the DC-SIGN and DC-SIGNR proteins and z. A peptide, a cyclic peptide, a polypeptide, an antibody or an antibody fragment may include. These compounds may be proteins or synthetic Act agents that have been designed so that they certain Mimic structures or epitopes on the inhibitor protein. Preferably is the compound is an antibody or an antibody fragment.

Of the Term "functionally equivalent Analog "also closes any analogue of DC-SIGN or DC-SIGNR, which by a change the amino acid sequence is obtained, for. By one or more amino acid deletions, substitutions or additions in a way that the Protein analogue the ability maintains, HCV, preferably the envelope glycoproteins from HCV, to bind. amino acid substitutions can z. B. made by point mutations of the amino acid sequence encoding DNA become. In one embodiment reserves the analogue the ability to bind HCV, but does not bind ICAM-3.

at the functional equivalent DC-SIGN or DC-SIGNR may be an analog or a Fragment of the DC-SIGN or DC-SIGNR act. The DC-SIGN or DC-SIGNR or the functional equivalent of which can be chemically modified, provided that that it's his ability to retain HCV, preferably the envelope glycoproteins of HCV.

The The present invention also discloses functional equivalents of such polypeptides and fragments thereof. Such functional equivalents can be at least 75% homologous to the native sequence. Such functional equivalents can also at least 80%, at least 85%, at least 90%, at least 95% or at least 100% homologous to the native sequence. at a functionally equivalent Fragment may be a fragment of the polypeptide which still binds to its targeting ligands. So z. B. a functionally equivalent Fragment of the E1 ectodomain a fragment which is a deletion of at least one amino acid on its aminoterminal end, at its carboxy-terminal end, internally or a combination of it and still at his Ligands on the for susceptible to HCV infection Cell binds.

The The present invention also discloses functionally equivalent Analogs of such polypeptides and polypeptide fragments. Such Have analogues an activity, which is analogous to that of the polypeptide or fragment. Such Analogues could through change the amino acid sequence obtained as by insertion, deletion or substitution of at least one amino acid. Such an analog would still bind to its ligand. So z. B. an E1 analogue still always on its ligand susceptible to HCV infection Bind cell. For amino acid substitutions they can be conservative substitutions. In such Conservative substitutions may be substitutions within of the following groups: (1) glycine and alanine; (2) Valine, Isoleucine and leucine; (3) aspartic acid and glutamic acid; (4) Asparagine and glutamine; (5) serine and threonine; (6) lysine and arginine; (7) phenylalanine and tyrosine. With such substitutions it can are also homologous substitutions, as well as within the following groups: (a) glycine, alanine, valine, leucine and isoleucine; (B) Phenylalanine, tyrosine and tryptophan; (c) lysine, arginine and histidine; (d) aspartic acid and glutamic acid; (e) asparagine and glutamine; (f) serine and threonine; (g) cysteine and methionine.

The functional equivalent can also be modified, as by a chemical modification, wherein however, it still binds to its corresponding ligand.

It is foreseen that such molecules in the preventive Therapy of HCV infection be useful be, because these molecules specifically bind to the virus and consequently the virus prevent in cells. As used herein, "specific bind that " functionally equivalent Analog high affinity for HCV or the HCV envelope glycoproteins but not for Control proteins. The specific binding can be done by means of a series by techniques such as ELISA, flow cytometry, Western Blotting or immunoprecipitation. Preferably, the functionally equivalent analog binds in nanomolar or picomolar concentrations specific to HCV or the HCV envelope glycoproteins.

The present application also discloses a compound which binds to DC-SIGN and / or DC-SIGNR for use in the diagnosis or therapy of HCV. Preferably, the compound binds in nanomolar or picomolar concentrations specifically to DC-SIGN and / or DC-SIGNR. such compounds can be used to prevent the virus from binding and infecting target cells. The compound includes, but is not limited to: an antibody, a carbohydrate, a small molecule, a peptide, a polypeptide and an oligopeptide.

The DC-SIGN protein, DC-SIGNR protein or the functional equivalent of which can be made by any suitable means, as for the skilled person will be apparent. To produce sufficient Amounts of DC-SIGN protein, the DC-SIGNR protein or functional equivalents thereof for use according to the present Invention can achieve expression in a suitable manner be that recombinant host cells containing the DC-SIGNR protein or a functional equivalent thereof cultured under suitable conditions. Preferably becomes the DC-SIGN or DC-SIGNR protein produced by recombinant means, by expression a coding nucleic acid molecule. systems for cloning and expression of a polypeptide in a variety different host cells are well known.

Becomes expressing it in recombinant form, the DC-SIGN protein, the DC-SIGNR protein or the functional equivalent preferably by expression from a coding nucleic acid in a Host cell generated. It can use any host cell be, depending on the individual requirements of a particular System. Suitable host cells include bacterial cells, mammalian cells, Plant cells, yeast and baculovirus systems. In the state of Technology available mammalian cells ovarian cells include expression of a heterologous polypeptide Chinese hamsters, HeLa cells, kidney cells from hamster babies and many others. Bacteria are due to the Ease with which bacteria are manipulated and bred can, also preferred hosts for the production of recombinant protein. A common one preferred bacterial host is E. coli.

The nucleic acids, Polypeptides and antibodies or any other agent / s described herein Other compounds described herein can be isolated and / or purified become. The person skilled in the art is able to isolate these and / or purify. Appropriate methods are used in any Laboratory manual, such as "Molecular Cloning" by Sambrook, Fritsch and Maniatis.

As The following are used throughout the specification standard abbreviations related to specific amino acids to denote: A = ala = alanine; R = arg = arginine; N = asn = asparagine; D = asp = aspartic acid; C = cys = cysteine; Q = gln = glutamine; E = glu = glutamic acid; G = gly = glycine; H = his = histidine; I = ile = isoleucine; L = Ieu = leucine; K = lys = lysine; M = met = methionine; F = phe = phenylalanine; P = per = proline; S = ser = serine; T = thr = threonine; W = trp = tryptophan; Y = tyr = tyrosine; and V = val = valine.

The present invention discloses a transgenic non-human animal comprising a transgene encoding the relevant polypeptide or a functional equivalent thereof. See, for example, B. U.S. Patent No. 6,025,539 ; IL-5 transgenic mouse; U.S. Patent No. 6,023,010 Transgenic non-human animals depleted in a mature lymphocytic cell type; U.S. Patent No. 6,018,098 , In vivo and in vitro model of cutaneous photoaging; U.S. Patent No. 6,018,097 , Transgenic mice express human insulin; U.S. Patent No. 6,008,434 , Growth differentiation factor-11 transgenic mice; U.S. Patent No. 6,002,066 , H2-M modified transgenic mice; U.S. Patent No. 5,994,618 , Growth differentiation facto-8 transgenic mice; U.S. Patent No. 5,986,171 , Method for examining neurovirulence of polio virus; U.S. Patent No. 5,981,830 Knockout mice and their progeny with a disrupted hepsin gene; U.S. Patent No. 5,981,829 , .DELTA.Nur77 transgenic mouse; U.S. Patent No. 5,936,138 Gene encoding mutant L3T4 protein which causes HIV infection and transgenic mouse expressing such protein; U.S. Patent No. 5,912,411 Mice transgenic for a tetracycline-inducible transcriptional activator; U.S. Patent No. 5,894,078 , Transgenic mouse expressing C-100 app.

The methods used to generate transgenic mice are well known to those skilled in the art. So z. For example, the handbook entitled "Manipulating the Mouse Embryo" by Brigid Hogan et al., (Ed., Cold Spring Harbor Laboratory), 1986. See, for example, Leder and Stewart, U.S. Patent No. 4,736,866 for methods of generating a transgenic mouse.

It has been known for some time that it is possible to genetic transformation a zygote (and the embryo and the mature organism which result from this), by the placement or insertion of exogenous genetic Material in the nucleus of the zygote or in any nuclear genetic Material, which ultimately forms part of the nucleus of the zygote forms. The genotype of the zygote and that resulting from a zygote Organism will include the genotype of the exogenous genetic material. In addition will the inclusion of exogenous genetic material in the zygote a phenotype expression of exogenous genetic material.

Of the Genotype of the exogenous genetic material becomes after cell division the zygote is expressed. However, phenotype expression, e.g. B. the Production of a protein product or products of the exogenous genetic material, or changes of the natural phenotype the zygote or organism, at the point of development of the Zygote or organism, during which the specific exogenous genetic material is active. Changes in the expression of the phenotype shut down an: an increase or decrease in expression or a change in the promotion and / or control of a phenotype, including the Addition of a new promoter and / or controller or the replacement of a existing promoter and / or controller of the phenotype.

The genetic transformation of various types of organisms is disclosed and described in detail U.S. Patent No. 4,873,191 , issued October 10, 1989. Genetic transformation of organisms can be used as an in vivo analysis of gene expression during differentiation, as well as in the elimination or amelioration of genetic diseases either by gene therapy or by the use of a transgenic non-human mammal as a model system of the human disease. This model system can be used to test putative drugs for their potential therapeutic benefit in humans.

The Exogenous genetic material can enter the nucleus of a mature ice to be placed. It is preferred that the egg is in a fertilized or activated (by parthenogenesis) state. To the addition of the exogenous genetic material becomes a haploider Set of chromosomes (such as a sperm cell or a polar body) added to the To allow formation of a zygote. It then allows the development of the zygote to an organism, as well as by implantation in a pseudopregnant female. Of the Resulting organism is in terms of integration exogenous genetic material. Will be a positive Determined integration, the organism can be used for in vivo analysis of gene expression be used, which is assumed to be with a specific genetic disorder.

The "transgenic not human animals " by creating "transgenes" in the germline of the non-human animal. It can embryonic target cells at various stages of development for the introduction of Transgenics are used. It will be according to the stage of development the embryonic target cell different procedures applied. The zygote is the most suitable target for microinjection. In mice reaches the male Pronucleus a size of about 20 microns in diameter, giving a reproducible injection from 1-2 pl DNA solution allows. The use of zygotes as a target for gene transfer has thus far a significant advantage in that the injected DNA in the most cases will be integrated into the host gene before the first cleavage (Brinster et al. (1985) Proc. Natl. Acad. Sci. USA 82, 4438-4442). As a result of these, all cells of the transgenic nonhuman animal become the carry integrated transgene. This is also becoming common in the efficient transgene transduction to the progeny of the transgene Founders reflect that 50% of the germ cells harbor the transgene become. The microinjection of zygotes is the preferred methods for integrating transgenes in the practical execution the invention.

It a retroviral infection can also be used to To introduce transgenes into a nonhuman animal. The one in development conceived non-human embryo can in vitro until the blastocyst stage be cultivated. While this time can the blastomers as targets for the retroviral infection serve (Jaenich, R. (1976) Proc. Natl. Acad. Sci. USA 73, 1260-1264). Efficient blastomere infection occurs by enzymatic treatment to remove the zona pellucida (Hogan et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA). At the introduction The viral vector system used in the transgene is typically a replication-deficient retrovirus containing the transgene bears (Jahner et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6927-6931; Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-6152). Transfection is easy and efficient through cultivation blastomeres on a monolayer of virus-producing cells (Van the putti, supra; Stewart et al. (1987) EMBO J. 6, 383-388). alternative In addition, the infection can be carried out at a later stage. It can Virus or virus producing cells injected into the blastozoel (Jahner, D. et al. (1982) Nature 298, 623-628). Most the founders become mosaic for the Being transgenic, since the integration only in a subset of cells which formed the transgenic non-human animal. Furthermore The founder can different retroviral at different positions in the genome Include insertions of the transgene, which are generally to isolate in the offspring. Moreover, it is also possible the Transgenic by intrauterine retroviral infection of the middle to introduce the gestation embryo into the germline if even with low efficiency (Jahner, D. et al. (1982) supra).

Around a third type of target cell for the introduction of transgenes is the embryonic stem cell (ES). ES cells are cultured from in vitro before implantation Embryos and fused with embryos (Evans, M.J. et al. (1981) Nature 292, 154-156; Bradley, M.O. et al. (1984) Nature 309, 255-258; Gossler et al. (1986) Proc. Natl. Acad. Sci. USA 83, 9065-9069; and Robertson et al. (1986) Nature 322, 445-448). By DNA transfection or by retrovirus-mediated transduction, transgenes can be effective in introduced the ES cells become. Such transformed ES cells can subsequently become infected with blastocysts be combined from a nonhuman animal. After that colonize the ES cells attach to the embryo and contribute to the germ line of the resulting chimeric Tiers at. For an overview see Jaenisch, R. (1988) Science 240, 1468-1474.

As As used herein, a "transgene" is a DNA sequence which is identified by human Intervention in the germline of a nonhuman animal, as by the previously described methods.

The Invention is illustrated in the following section "Experimental Details". These experimental details are intended to help understand the Invention; however, it is not intended that they be the invention as in the following claims limited in any way, and they should not be interpreted to that effect.

EXPERIMENTAL DETAILS

First series of experiments

 Structure glycoprotein E2 binding hepatitis C virus to human C-type lectins, DC-SIGN and DC-SIGNR

Summary

DC-SIGN, a human C-type lectin, is found on the surface of dendritic cells (DC) expressed, and one in the highest Dimensions homologous Protein, DC-SIGNR, is present in high concentrations on sinusoidal To find endothelial cells of the liver and lymph nodes. These molecules bind HIV envelope glycoprotein, gp120, and allow viral transmission in trans by attachment to DC. For HCV-E2 is the functional equivalent of HIV gp120 and it contains abundant oligosaccharides of the mannose-rich type, which on lectin molecules can bind DC-SIGN and DC-SIGNR. To verify this hypothesis, DC-SIGN or DC-SIGNR expressing HeLa cell lines and it became the binding evaluated on E2 protein and HCV virions. Using a fluorometric Bead assays were first demonstrated to be purified E2 at DC-SIGN and DC-SIGNR binds and that these interactions are due to a monoclonal antibody against DC-SIGN / DC-SIGNR additionally to mannan and calcium chelators. Based on these Experiments it looks like after that DC-SIGN and DC-SIGNR as co-receptors for the Attachment of HCV act and that their expression on DC as well significant effects on the endothelium of the liver and placenta has an HCV disease.

Materials and methods

Plasmids and cells

The Plasmids pcDNA3-DC-SIGN and pcDNA3-DC-SIGNR (article # 5444 resp. 6749, AIDS Research and Reference Reagent Program, Rockville, MD USA) were prepared using a lipid formulation (Effectene, Qiagen, Valencia, CA, USA) according to the above Manufacturer's protocol transfected into HeLa cells. Two Days after transfection, the cells were treated with standard growth media (Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), penicillin / streptomycin (Life Technologies, Carlsbad, CA, USA) and L-glutamine (Life Technologies) supplemented with 600 μg / ml geneticin (Life Technologies), treated. After two weeks, survivors were Cell colonies selected, expanded and using monoclonal Antibody, which DC-SIGN (507D), L-SIGN (612X) or both DC and L-SIGN (604L), screened by flow cytometry. The transfected HeLa cell lines were routinely used in DMEM added with 10% FBS, penicillin / streptomycin, L-glutamine with geneticin (600 μg / ml). The growing cells were harvested using a cell dissociation solution (Sigma, St. Louis, MO, USA) for divided a conservation culture.

antibody

• Bei Anti-E2-mAb-HCM-091-a-5 (Klon 4F6/2) von Austral Biologicals (San Ramon, CA, USA) handelt es sich um einen Maus-IgG1-mAb, welcher mit dem linearen Epitop in E2 und mit Serum aus HCV-seropositiven Donoren reagiert. Bei H31, H33, H44, H48, H53, H55, H60, H61 handelt es sich um anti-HCV-E2-Maus-mAbs (von Dr. Jean Dubuisson, Institut Pasteur de Lille, Frankreich), welche mit Konformationsepitopen (Deleersnyder et al., J. Virol. 71, 697-704 (1997), Flint et al., J. Virol. 73, 6782-6790 (1999)) kreuzreagieren.
• Bei 120507 (507D) von BD Pharmingen (San Diego, CA, USA) handelt es sich um ein DC-SIGN-spezifisches, auf die Lektin-Bindungsdomäne gerichtetes, konformationsabhängiges, murines IgG2b. 507D blockiert SIV- und HIV-Infektion sowie die ICAM-3-Adhäsion (Jameson et al., J. Virol. 76, 1866-1875 (2002), Wu et al., J. Virol. 76, 5905-5914 (2002)).
• Bei 120604 (6041) von BD Pharmingen (San Diego, CA, USA) handelt es sich um ein DC-SIGNR-spezifisches, auf die Lektin-Bindungsdomäne gerichtetes, konformationsabhängiges, murines IgG2b. 604L blockiert die Bindung an SIV und HIV nicht und zeigt nur geringe oder gar keine Blockierung der ICAM-3-Adhäsion (Jameson et al., J. Virol. 76, 1866-1875 (2002), Wu et al., J. Virol. 76, 5905-5914 (2002)).
• Bei 120612 (612X) von BD Pharmingen (San Diego, CA, USA) handelt es sich um ein murines IgG2a, welches die Lektin-Bindungsdomäne von sowohl DC-SIGN als auch DC-SIGNR erkennt. 612X blockiert ICAM-3-Adhäsion und HIV-Infektion (Jameson et al., J. Virol. 76, 1866-1875 (2002), Wu et al., J. Virol. 76, 5905-5914 (2002)).
• Bei DC4 (Artikel # 5442, AIDS Research and Reference Reagent Program, Rockville, MD, USA) handelt es sich um ein murines IgG1, welches sowohl DC-SIGN als auch DC-SIGNR erkennt, und zwar über die Neck- oder Repeat-Region und nicht über die Lektin bindende Domäne. DC4 blockiert ICAM-3-Bindung oder SIV-Transmission nicht (Baribaud et al., J. Virol., 10281-10289 (2001)).
• Bei DC28 (Artikel # 5443, AIDS Research and Reference Reagent Program, Rockville, MD, USA) handelt es sich um ein murines IgG2a, welches sowohl DC-SIGN als auch DC-SIGNR erkennt, und zwar über die Neck- oder Repeat-Region und nicht über die Lektin bindende Domäne. DC28 blockiert ICAM-3-Bindung oder SIV-Transmission nicht (Baribaud et al., J. Virol., 10281-10289 (2001)).

The following mAbs were used:

• Austral Biologicals (San Ramon, Calif., USA) anti-E2 mAb HCM-091-a-5 (clone 4F6 / 2) is a murine IgG1 mAb which interacts with the linear epitope in E2 and reacted with serum from HCV seropositive donors. H31, H33, H44, H48, H53, H55, H60, H61 are anti-HCV E2 mouse mAbs (from Dr. Jean Dubuisson, Institut Pasteur de Lille, France), which are infected with conformational epitopes (Deleersnyder et al., J. Virol. 71, 697-704 (1997), Flint et al., J. Virol. 73, 6782-6790 (1999)).
• BD Pharmingen 120507 (507D) (San Diego, CA) is a DC-SIGN-specific conformation-dependent murine IgG2b targeting the lectin-binding domain. 507D blocks SIV and HIV infection as well as ICAM-3 adhesion (Jameson et al., J. Virol., 76, 1866-1875 (2002), Wu et al., J. Virol., 76, 5905-5914 (2002 )).
• BD Pharmingen 120604 (6041) (San Diego, CA, USA) is a DC-SIGNR specific conformationally-dependent murine IgG2b directed to the lectin binding domain. 604L does not block binding to SIV and HIV and shows little or no blocking of ICAM-3 adhesion (Jameson et al., J. Virol. 76, 1866-1875 (2002), Wu et al., J. Virol 76, 5905-5914 (2002)).
• BD Pharmingen's 120612 (612X) (San Diego, CA) is a murine IgG2a that recognizes the lectin binding domain of both DC-SIGN and DC-SIGNR. 612X blocks ICAM-3 adhesion and HIV infection (Jameson et al., J. Virol., 76, 1866-1875 (2002), Wu et al., J. Virol., 76, 5905-5914 (2002)).
• DC4 (Article # 5442, AIDS Research and Reference Reagent Program, Rockville, Md.) Is a murine IgG1 that recognizes both DC-SIGN and DC-SIGNR through the neck or repeat region and not via the lectin binding domain. DC4 does not block ICAM-3 binding or SIV transmission (Baribaud et al., J. Virol., 10281-10289 (2001)).
• DC28 (Article # 5443, AIDS Research and Reference Reagent Program, Rockville, MD, USA) is a murine IgG2a that recognizes both DC-SIGN and DC-SIGNR through the neck or repeat region and not via the lectin binding domain. DC28 does not block ICAM-3 binding or SIV transmission (Baribaud et al., J. Virol., 10281-10289 (2001)).

immunofluorescence

The Cells were incubated in PBS / 0.5% BSA at 4 ° C for 30 minutes with primary mAbs colored and the addition of isotype-specific FITC-conjugated secondary mAbs (Anti-mouse FITC, BD Pharmingen, San Diego, CA, USA) for more 30 minutes at 4 ° C washed. After washing, the cells were harvested using a FACScan (Becton Dickinson, Mountain View, CA, USA) by flow cytometry analyzed. For the establishment of quadrant positions, isotype-specific Controls included.

Preparation of fluorescent HCV-E2 beads

Carboxylatmodifizierte were labeled with FluoSpheres ^® -NeutrAvidin ^TM microspheres (505/515 nm, 1.0 microns; Molecular Probes, Eugene, OR, USA) coated with HCV E2 envelope glycoprotein as described for ICAM-1 beads (Geijtenbeek et al., Blond 94, 754-764 (1999)). NeutrAvidin ^™ coated beads were sonicated briefly and washed in PBS / BSA (0.5%). The beads were probed with biotinylated, Sp-conjugated AffiniPure-F (ab ') ₂ goat anti-mouse IgG F (ab') ₂ fragment (6 μg / ml in PBS / BSA, 0.5%). ) (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) for 2 hours at 37 ° C. After washing, the beads were incubated at 4 ° C overnight with murine anti-E2 antibodies (6 μg / ml in PBS / BSA, 0.5%). The beads were washed and incubated with 250 ng / ml purified HCV-E2 prepared in CHO cells (Accurate Chemicals, NY, USA) overnight at 4 ° C. The identity of E2 protein was confirmed by Western blot analysis with anti-E2 mAbs (data not shown).

Adhesion assay for fluorescent Beads

This was done as described by Geijtenbeek et al. (Blond 94, 754-764 (1999)), with modifications. The cells were removed from the culture by cell dissociation solution (Sigma) for 5 minutes at 37 ° C and three times in adhesion buffer (20 mM Tris-HCl, [pH 8.0], 150 mM NaCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ and 0.5% BSA). The cells were resuspended for 30 minutes at 4 ° C at a final concentration of 5 x 10 ⁶ cells / ml in adhesion buffer to recharge the Ca ²⁺ levels. The cells (5 x 10 ⁵ ) were preincubated with mannan (20 μg / ml; Sigma), antibodies (0.1-10 μg / ml), EDTA (5 mM) or EGTA (5 mM) for 10 minutes at room temperature. HCV-E2 coated fluorescent beads (20 beads / cell) were added and the suspension was incubated for 30 minutes at 37 ° C. The adhesion was through the Measurement of the percentage of cells that bound fluorescent beads determined by flow cytometry using a FACScan (Becton Dickinson, Oxnard, CA, USA).

Results

To examine whether DC-SIGNR and DC-SIGNR are receptors for HCV-E2, stable HeLa cell lines were prepared by transfecting cDNAs encoding either DC-SIGN or DC-SIGNR. Selected clones of these cells HeLa-DC-SIGN and HeLa-DC-SIGNR) were screened using a panel of anti-DC-SIGN or anti-DC SIGNR-specific antibodies which have been reported to be reactive with human tissues ( 4 and Table 1, below), a flow cytometric analysis was performed. Large amounts of DC-SIGN and DC-SIGNR molecules were expressed on the surfaces of the respective cell lines. HeLa parent cells stained with none of the anti-DC SIGN or anti-DC SIGNR antibodies. Antibody clone specificity % positive cells (mean fluorescence intensity) HeLa DC-SIGN HeLa DC SIGNR HeLa cells parents 507 (D) DC-SIGN 90.6 (81.1) 3,5 (17,1) 0.8 (7.8) 612 (X) DC-SIGN, DC-SIGNR 67.3 (46.6) 94.8 (102.1) 0.3 (8.1) 604 (L) DC-SIGN 1.1 (14.2) 96.0 (128.9) 0.1 (8.6) Isotype control None 1.0 (16.9) 2.8 (13.7) 0.1 (7.9) Table 1. Surface expression of DC-SIGN and DC-SIGNR in stable HeLa-DC-SIGN and HeLa-DC-SIGNR cell lines.

To determine whether DC-SIGN and DC-SIGNR bind to HCV-E2 envelope glycoprotein, a flow cytometric adhesion assay (Geijtenbeek et al., Blood 94, 754-764 (1999)) was adapted. HCV E2 protein produced in CHO cells was captured on fluorescent beads using a panel of anti-E2 mAbs, which was present in a ratio of 20 beads per cell with DC-SIGN and DC-SIGNR-HeLa Cells were incubated. The HCV-E2 coated beads bound efficiently to both cell types and the binding was performed by mannan ( 5 ) and EDTA or EGTA (data not shown) are efficiently inhibited. Low levels of background adhesion were observed in the untransfected HeLa parental cell line expressing no DC-SIGN or DC-SIGNR. Binding levels depended on the anti-E2 mAb used for coating, but the course was more similar for DC-SIGN and DC-SIGNR cells. Only antibody and E2 protein-conjugated beads did not bind to cells (data not shown).

The panel of anti-DC SIGN and anti-DC SIGNR mAbs was tested for their effect on E2: DC-SIGN / DC-SIGNR adhesion in the fluorescent bead binding assay ( 6 ). The binding of E2 to DC-SIGNR was inhibited by DC4, an antibody that recognizes both DC-SIGN and DC-SIGNR; however, binding to DC-SIGN was only moderately inhibited by a subset of mAbs in the panel, suggesting that E2 could interact with different sites on these molecules. DC4 does not inhibit the attachment of lentivirus or the binding of ICAM-3 to DC-SIGN and DC-SIGNR, and thus could be a novel HCV-specific inhibitor.

discussion

It Thus, it has been shown that HCV-E2 envelope glycoprotein with DC-SIGN and DC-SIGNR interacts and that mannan, calcium chelators, and an anti-DC SIGN / DC SIGNR mAb make this interaction inhibited. These findings are new and the expression of these potential HCV receptors on DC and the endothelium has significant Impact on the viral life cycle. DC-SIGN expressed on DC could be similar like HIV HCV in trans to susceptible Transfer cells and the expression of DC-SIGNR in the liver and placenta could be the control viral tropism and the subsequent pathogenesis.

Sinusoidal endothelial cells of the liver are in constant contact with passing leukocytes and can catch viruses, apoptotic cells and antigens from the blood and promote trans-infection of target cells. It is therefore possible that DC-SIGNR promotes infection of these cells, thereby establishing a reservoir for the production of new virus for transmission to hepatocytes. A similar Me Mechanism could affect the vertical transmission of HCV across the terminal placenta, a tissue containing high levels of DC-SIGNR. Inhibition of these interactions represents therapeutic and prophylactic strategies in HCV disease. The role of DC-SIGNR and DC-SIGN as binding molecules controlling the trafficking of HCV and its localization in the liver remains to be fully elucidated; however, their interactions with HCV-E2 represent novel targets for therapeutic intervention.

Second series of experiments

Summary

It becomes an inhibitor of attachment of HCV to DC-SIGN and / or DC-SIGNR described the binding of HCV-positive sera or purified Virions from the assay described below override puts. This inhibitor can be used with virus or the receptor or interact with both.

Serum binding to cells

HeLa cell lines (HeLa-DC-SIGN, HeLa, DC-SIGNR) or parental HeLa cells are cultured overnight in DMEM containing 10% FBS in a 24-well plate at 1 x 10 ⁵ cells / well. The following day, the cells are washed once with adhesion buffer and then blocked with adhesion buffer containing 10% heat-inactivated goat serum for 20 minutes at 37 ° C. The cells are washed once with adhesion buffer and one or more inhibitors in adhesion buffer are added in half of the wells for 1 hour. Previously, ten μL of either HCV RNA + (virus positive) or HCV RNA (virus negative) serum is diluted to a final volume of 200 μL and added to the wells. One or more inhibitors may also be added to aliquots of the sera for 1 hour to allow interaction with virus. The virus is allowed to bind to cells for 1 hour at 37 ° C, stirring gently every 15 minutes. Finally, the serum is removed and the cells are washed five times with adhesion buffer.

Viral RNA extraction

viral RNA is generated using a QIAmp Viral RNA Mini Spin Kit (Qiagen) extracted with modifications from cells. In short, it will be two extractions with 280 μL Lysis buffer added per well and transferred to a 1.7 mL vessel. The empty plate comes with 140 μL Dulbecco's phosphate buffered Saline washed with calcium and magnesium and pooled in the same vessel. RNA extraction and binding to spin columns are according to the guidelines of the manufacturer. Following washing with washing buffer is the DNA on the column by treatment with RNase-free DNase (Qiagen) using removed from the manufacturer's guidelines. The RNA is washed, using 30 μL or 40 μL Elution buffer eluted in two steps and the eluates are combined.

HCV-specific RT-PCR

Half a nmol of the primer RJD-5 is combined with 0.5 μL of extracted RNA in a final volume of 6 μL. The samples are heated for 10 minutes at 70 ° C and then cooled to 4 ° C using the GeneAmp PCR system (Perkin Elmer). In a 10 μL reaction mixture, the preheated template is combined with 1 × first strand buffer, 10 mM DTT, 5 mM deoxyribonucleoside triphosphates (dNTPs) and 7.5 U ThermoScript (Invitrogen), at 58 ° C for 50 minutes, then at 85 ° C for 5 Incubated for a few minutes before being cooled to 4 ° C. From this RT reaction 5 μL are used as template for a PCR in a 50 μL reaction containing 1 × high fidelity PCR buffer, 2 mM MgSO ₄ , 2 mM dNTPs, 50 pmol primers RJD-1 and RJD-5 and 1 , 25 U Platinum Taq High Fidelity DNA Polymerase (Invitrogen). PCR amplification is carried out using the method of Young et al. (Young, KK, Resnik, RM, Myers, TW, Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay J. Clin. Microbiol., April 3, 1993, 31 (4): 882-6 ).

Blotting of the RT-PCR products

10 μL of each RT-PCR reaction is resolved on a 1% agarose gel containing a biotinylated DNA ladder (NEB). According to the method described in Sambrook, Fritsch and Maniatis (Sambrook, J., Fritsch, EF and Maniatis, T. in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, USA, Vol. 1, 2, 3 (1989) ), the gel is capillary to a proton nitrocellulose membrane (type BA-85, Schleicher and Schull) blotted. The following day the DNA is at 2400 J / m ² using a Stratalinker (Stratagene) cross-linked to the membrane and dried for at least 1 hour at room temperature.

Hybridization for detection from HCV

Of the Blot is for 4 hours at 63 ° C incubated in prehybridization solution. The prehybridization solution contains 5x Denhardt's [0.2% (w / v) fatty acid-free BSA (JRH Biosciences), 0.2% (w / v) polyvinylpyrrolidonepolyvinyl (PVP, Sigma), 0.2% (w / v) Fikoll-400 (Sigma)], 6 x SSC (0.9 M NaCl, 90 mM sodium citrate, pH 7.4), 0.5% (w / v) sodium dodecyl sulfate (SDS, Promega) and 0.1 mg / mL herring sperm DNA (Invitrogen). Following the incubation 1 pmol / mL of primer RJD-6 or RJD-7 is added to the prehybridization solution, to the hybridization solution and incubate at 63 ° C overnight. The following morning, the blot is run twice for 5 minutes in washing buffer [2 × SSC, 0.1% (w / v) SDS] at room temperature, twice in wash buffer for 15 minutes at 63 ° C and washed once more in wash buffer at room temperature. Of the Blot is then used once for 5 minutes in PEST [Dulbecco's phosphate-buffered saline without calcium and magnesium, 0.05% (v / v) Tween-20]. The blot is then left at room temperature for 45 minutes incubated with streptavidin-HRP (Amersham) at 1/1500 in PEST. Of the Blot is then washed twice quickly, then three times for 15 minutes in PEST. Using Western Lightning (NEN / Perkin Elmer) and Kodakfilm the blot is developed. An HCV RAN positive signal is transmitted through illustrates a specific band of 243 base pairs. The intensity the 243 base pair band is in presence and absence compared to inhibitor; shows a reduction in intensity inhibition of HCV binding.

Third series of experiments

Summary

It is a new, improved method for the detection of HCV in Samples from humans (blood, serum, plasma, tissue, amniotic fluid, etc.), which is that in the example below operated assays. In this procedure, the samples in the cell binding assay (SIGN assay) for attachment tested on HeLa cells expressing DC-SIGN and / or DC-SIGNR. The standard cell-free Assay is used as a control at different sample concentrations used to limit the detection and linearity of the SIGN assay to determine. This test gives additional quantitative information on the viral load of HCV (eg increased sensitivity in detection the presence of HCV in a biological sample), in addition to Qualitative properties (binding of DC-SIGN or DC-SIGNR) of the virus present in the sample. This assay provides novel information ready, which for the use of receptors and the distribution of viral Quasi-species (eg. Pathogenic phenotypes) are relevant and therefore in the monitoring of clinical progression a disease of benefit.

Sample binding to cells

HeLa cell lines (HeLa-DC-SIGN, HeLa, DC-SIGNR) or parental HeLa cells are cultured overnight in DMEM containing 10% FBS in a 24-well plate at 1 x 10 ⁵ cells / well. The following day, the cells are washed once with adhesion buffer and then blocked with adhesion buffer containing 10% heat-inactivated goat serum for 20 minutes at 37 ° C. The cells are washed once with adhesion buffer. A fixed volume (eg, 10-1,000 μL) of either HCV RNA + (virus positive) or HCV RNA (virus negative) serum or other samples (plasma, tissue extracts, etc.) in Ad Dilute the buffer to a final volume of 200 μL and prepare a series of 10-fold serial dilutions. These suspensions are added to the wells for 1 hour to allow interaction with virus and incubated for 1 hour at 37 ° C with gentle stirring every 15 minutes. Finally, the sample is removed and the cells are washed five times with adhesion buffer. To determine the limit of detection and linearity of the assay, aliquots of the same samples without cell binding (cell-free samples) are used in subsequent steps as discussed below

Viral RNA extraction

viral RNA is generated using a QIAmp Viral RNA Mini Spin Kit (Qiagen) extracted with modifications from cells. In short, it will be two extractions with 280 μL Lysis buffer added per well and transferred to a 1.7 mL vessel. The empty plate comes with 140 μL Dulbecco's phosphate buffered Saline washed with calcium and magnesium and pooled in the same vessel. RNA extraction and binding to spin columns are according to the guidelines of the manufacturer. Following washing with washing buffer is the DNA on the column by treatment with RNase-free DNase (Qiagen) using removed from the manufacturer's guidelines. The RNA is washed, using 30 μL or 40 μL Elution buffer eluted in two steps and the eluates are combined.

HCV-specific RT-PCR

Half a nmol of the primer RJD-5 is combined with 0.5 μL of extracted RNA in a final volume of 6 μL. The samples are heated for 10 minutes at 70 ° C and then cooled to 4 ° C using the GeneAmp PCR system (Perkin Elmer). In a 10 μL reaction mixture, the preheated template is combined with 1 × first strand buffer, 10 mM DTT, 5 mM deoxyribonucleoside triphosphates (dNTPs) and 7.5 U ThermoScript (Invitrogen), at 58 ° C for 50 minutes, then at 85 ° C for 5 Incubated for a few minutes before being cooled to 4 ° C. From this RT reaction 5 μL are used as template for a PCR in a 50 μL reaction containing 1 × high fidelity PCR buffer, 2 mM MgSO ₄ , 2 mM dNTPs, 50 pmol primers RJD-1 and RJD-5 and 1 , 25 U Platinum Taq High Fidelity DNA Polymerase (Invitrogen). PCR amplification is carried out using the method of Young et al. (Young, KK, Resnik, RM, Myers, TW, Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay J. Clin. Microbiol., April 1993, 31 (4): 882-6).

Blotting of the RT-PCR products

10 μL of each RT-PCR reaction is resolved on a 1% agarose gel containing a biotinylated DNA ladder (NEB). According to the method described in Sambrook, Fritsch and Maniatis (Sambrook, J., Fritsch, EF and Maniatis, T. in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, USA, Vol. 1, 2, 3 (1989) ), the gel is capillary blotted onto a Protran nitrocellulose membrane (type BA-85, Schleicher and Schull). The following day the DNA is at 2400 J / m ² using a Stratalinker (Stratagene) cross-linked to the membrane and dried for at least 1 hour at room temperature.

Hybridization for detection from HCV

The blot is incubated for 4 hours at 63 ° C in prehybridization solution. The prehybridization solution contains 5 × Denhardt's [0.2% (w / v)] fatty acid-free BSA (JRH Biosciences), 0.2% (w / v) polyvinylpyrrolidone polyvinyl (PVP, Sigma), 0.2% (w / v) ficoll 400 (Sigma)], 6x SSC (0.9 M NaCl, 90 mM sodium citrate, pH 7.4), 0.5% (w / v) sodium dodecyl sulfate (SDS, Promega), and 0.1 mg / mL herring sperm. DNA (Invitrogen). Following incubation, 1 pmol / mL of primer RJD-6 or RJD-7 is added to the prehybridization solution to prepare the hybridization solution and incubated at 63 ° C overnight. The following morning, the blot is washed twice for 5 minutes in washing buffer [2 × SSC, 0.1% (w / v) SDS] at room temperature, twice for 15 minutes in washing buffer at 63 ° C. and once again in washing buffer at room temperature , The blot is then washed once for 5 minutes in PEST [Dulbecco's phosphate buffered saline without calcium and magnesium, 0.05% (v / v) Tween-20]. The blot is then incubated for 45 minutes at room temperature with streptavidin-HRP (Amersham) at 1/1500 in PEST. The blot is then washed twice quickly, then three times for 15 minutes in PEST. Using Western Lightning (NEN / Perkin Elmer) and Kodakfilm, the blot is developed. an HCV RAN positive signal is exemplified by a specific band of 243 base pairs. The intensity of the 243 base pair band is compared between identical samples in the cell binding (SIGN) assay and the cell-free assay for quantitative and qualitative differences. The difference between the signal intensities, wel Observed in the DC-SIGN and DC-SIGNR assays, it provides information important to the use and tropism of HCV receptor and ultimately to clinical progression.

It are numerous embodiments of the assay described above. So z. On HeLa-DC-SIGN- and / or HeLa DC SIGNR cells captured HCV using conventional Display values, as by Western blot analysis of HCV proteins using antibodies to viral proteins. In a further embodiment can purified DC-SIGN and / or DC-SIGNR proteins using common technologies a surface be immobilized, such as on a plate or on a bead, and used to capture HCV from patient samples and concentrate. The amount of HCV can be determined by measuring the number of the viral genomes by RT-PCR methods as described Western blot analysis of viral proteins, by ELISA or by other standard methodologies which are well known to those skilled in the art.

Of the One skilled in the art will readily recognize that those discussed herein specific procedures and results for illustrative purposes only serve the invention, which more fully described in the following claims will be.

references

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SEQUENCE LISTING

Claims (42)

Use of an antibody or a part thereof in an amount sufficient to bind hepatitis C virus (HCV) envelope glycoprotein to inhibit a DC-SIGNR protein present on the surface of a Cell present is for the manufacture of a medicament for administration to a Subject for inhibiting HCV infection of cells of this subject, which are vulnerable for one Are HCV infection. Use of an antibody or a part thereof in an amount sufficient to bind hepatitis C virus (HCV) envelope glycoprotein to inhibit a DC-SIGNR protein present on the surface of a Cell present is for the manufacture of a medicament for administration to a Subject for inhibiting HCV infection of a target cell of the Subject whose susceptibility for one HCV infection is increased, when HCV binds to a DC-SIGNR protein expressing cell. Use according to claim 1 or 2, wherein it is at the for an HCV infection vulnerable Cell is a liver hepatocyte cell. Use according to claim 1 or 2, wherein it is at the DC-SIGNR protein expressing cell is a liver-sinusoidal cell. Use according to any one of claims 1 to 4, wherein it is in the antibody or the part thereof is a monoclonal antibody. Use according to any one of claims 1 to 4, wherein it is in the antibody or the part thereof is a polyclonal antibody. Use according to any one of claims 1 to 4, wherein it is in the antibody or part of which is a humanized antibody. Use according to any one of claims 1 to 4, wherein it is in the antibody or part of it is a chimeric antibody. Use according to any one of claims 1 to 4, wherein it is in the antibody or part of it is a part of the antibody. Use according to claim 9, wherein the part of the antibody is a light chain of the antibody includes. Use according to claim 9, wherein the part of the antibody is a heavy chain of the antibody includes. Use according to claim 9, wherein the part of the antibody is a Fab fragment comprises. Use according to claim 9, wherein the part of the antibody comprises an F (ab ') ₂ fragment. Use according to claim 9, wherein the part of the antibody is a Fd fragment comprises. Use according to claim 9, wherein the part of the antibody is a Includes Fv fragment. Use according to claim 9, wherein the part of the antibody is a variable domain of the antibody. Use according to claim 9, wherein the part of the antibody is a or multiple CDR domains of the antibody includes. Use according to any one of claims 1 to 17, wherein it is in the HCV envelope glycoprotein an E1-HCV envelope glycoprotein is. Use according to any one of claims 1 to 17, wherein it is in the HCV envelope glycoprotein an HCV E2 envelope glycoprotein is. Use according to any one of claims 1 to 19, wherein the antibody or the part of it orally, intravenously, subcutaneous, intramuscular, topically or by liposome-mediated delivery is administered. Use according to any one of claims 1 to 20, wherein it is in the subject around a human, a primate, an equines, an ovines, an avianes, a bovines, a porcines, a canines, a feline or a murine subject. Use according to claim 21, wherein the effective Amount of the antibody or part thereof between 1 mg and 50 mg per kg of body weight of the subject. Use according to claim 22, wherein the effective Amount of the antibody or part thereof between 2 mg and 40 mg per kg of body weight of the subject. Use according to claim 23, wherein the effective Amount of the antibody or part thereof between 3 mg and 30 mg per kg of body weight of the subject. Use according to claim 24, wherein the effective Amount of the antibody or part thereof between 4 mg and 20 mg per kg of body weight of the subject. Use according to claim 25, wherein the effective Amount of the antibody or part thereof between 5 mg and 10 mg per kg of body weight of the subject. Use according to claim 19, wherein the antibody or the part of it is administered at least once a day. Use according to claim 27, wherein the antibody or the part of it daily is administered. Use according to claim 19, wherein the antibody or the part of it is administered every other day. Use according to claim 19, wherein the antibody or the part of it is administered every 6 to 8 days. Use according to claim 30, wherein the antibody or the part of it weekly is administered. A method for determining if a connection is capable of an HCV infection to inhibit a cell comprising: a) Immobilize an HCV envelope glycoprotein on a solid support; b) Contacting the immobilized HCV envelope glycoprotein with a sufficient Amount of detectable DC-SIGNR protein around all binding sites for the Saturate DC-SIGNR protein on the immobilized HCV envelope glycoprotein, under conditions which immobilized the binding of the DC-SIGNR protein to the HCV envelope glycoprotein permit so that a complex is formed; c) Remove unbound DC-SIGNR protein; d) contact the complex with the compound; e) Determine if any DC-SIGNR protein is displaced from the complex, with a displacement of the DC SIGNR protein from the complex indicates that the compound is attached the HCV envelope glycoprotein binds, to thereby determine that the connection is a is one that is capable of HCV infection of the cell to inhibit. A method for determining if a connection is capable of an HCV infection to inhibit a cell comprising: a) Immobilize a DC-SIGNR protein on a solid support; b) bring into contact of the immobilized DC-SIGNR protein with a sufficient amount on detectable HCV envelope glycoprotein, around all binding sites for the HCV envelope glycoprotein saturate on the immobilized DC-SIGNR protein, under conditions which binds the immobilized DC-SIGNR protein to the HCV envelope glycoprotein permit so that a complex is formed; c) Remove unbound HCV envelope glycoprotein; d) Contacting the complex with the compound; e) determining whether any HCV envelope glycoprotein displaced from the complex being, being a repression of the HCV envelope glycoprotein indicates from the complex that the compound binds to the DC-SIGNR protein, to thereby determine that the connection is a is one that is capable of HCV infection of the cell to inhibit. A method for determining if a connection is capable of an HCV infection to inhibit a cell comprising: a) bring into contact an HCV envelope glycoprotein with a sufficient amount of detectable DC-SIGNR protein, around all binding sites for to saturate the DC-SIGNR protein on the immobilized HCV envelope glycoprotein, under conditions involving binding of the DC-SIGNR protein to the Allow HCV envelope glycoprotein, so that a complex is formed; b) removal of unbound DC SIGNR protein; c) measuring the amount of DC-SIGNR protein, which bind to the HCV envelope glycoprotein is bound in the complex; d) contacting the complex with the compound, thus allowing the DC-SIGNR protein from the complex displace; e) Measure the amount of DC-SIGNR protein present in the presence the compound is bound to the compound; and f) Compare the amount of DC-SIGNR protein produced in step e) to the HCV envelope glycoprotein is bound, with the measured in step c) amount, wherein a reduced amount measured in step e) indicates that the compound to the HCV envelope glycoprotein binds to thereby identify the compound as such which is able to inhibit the HCV infection of a cell. A method for determining whether a compound is capable of HCV infection of a cell which comprises: a) immobilizing an HCV envelope glycoprotein on a solid support; b) contacting the immobilized HCV envelope glycoprotein with the compound as well as detectable DC-SIGNR protein under conditions which allow binding of the DC-SIGNR protein to the immobilized HCV envelope glycoprotein in the absence of the compound to form a complex ; c) removing unbound DC-SIGNR protein; d) comparing the amount of detectable DC-SIGNR protein bound in the presence of the compound to the immobilized HCV envelope glycoprotein in the complex with the amount of detectable DC-SIGNR protein which, in the absence of the compound, binds to the immobilized HCV envelope glycoprotein binds; e) wherein a reduced amount of DC-SIGNR protein measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein to thereby determine that it is the compound is one which is capable of inhibiting HCV infection of the cell. The method of claim 35, wherein the amount of the detectable DC-SIGNR protein sufficient to bind all DC-SIGNR protein binding sites on the HCV envelope glycoprotein saturate. A method for determining if a connection is capable of an HCV infection to inhibit a cell comprising: a) immobilize a DC-SIGNR protein on a solid support; b) bring into contact of the immobilized DC-SIGNR protein with the compound as well as with detectable HCV envelope glycoprotein under conditions involving binding of the immobilized DC-SIGNR protein to the HCV envelope glycoprotein allow in the absence of the compound, leaving a complex is formed; c) removing unbound HCV envelope glycoprotein; d) Comparing the amount of detectable HCV envelope glycoprotein present in the presence of the compound on the immobilized DC-SIGNR protein bound in the complex, with the amount of detectable HCV envelope glycoprotein, which is immobilized in the absence of the compound DC SIGNR protein binds; e) wherein a reduced amount of HCV envelope glycoprotein, which is measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein binds to thereby determine that it is in the connection is one that is capable of causing a HCV infection of the cell to inhibit. The method of claim 37, wherein the amount of the detectable HCV envelope glycoprotein is sufficient to all the binding sites for the HCV envelope glycoprotein saturate on the DC-SIGNR protein. A method for determining if a connection is capable of an HCV infection to inhibit a cell comprising: a) bring into contact an HCV envelope glycoprotein with the compound as well as detectable DC-SIGNR protein under Conditions involving binding of the DC-SIGNR protein to the HCV envelope glycoprotein in the absence allow the compound to form a complex; b) Removing unbound DC-SIGNR protein; c) Compare the amount of detectable DC-SIGNR protein present in the presence the connection to the HCV envelope glycoprotein bound in the complex, with the amount of detectable DC-SIGNR protein, which in the absence of the compound to the HCV envelope glycoprotein binds; d) wherein a reduced amount of DC-SIGNR protein, which is measured in the presence of the compound indicates that the compound binds to the HCV envelope glycoprotein or to the DC-SIGNR protein binds to thereby determine that it is in the connection one that is capable of causing HCV infection to inhibit the cell. The method of claim 39, wherein the amount of detectable DC-SIGNR protein sufficient to bind all DC-SIGNR protein binding sites on the HCV envelope glycoprotein saturate. A method according to any one of claims 32, 34, 35 or 39, wherein the detectable DC-SIGNR protein with a detectable marker is marked. The method of claim 33 or 37, wherein the detectable HCV envelope glycoprotein is labeled with a detectable marker.